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Antibody structure, instability, and formulation

MINIREVIEWAntibody Structure,Instability,and FormulationWEI WANG,SATISH SINGH,DAVID L.ZENG,KEVIN KING,SANDEEP NEMAPﬁzer,Inc.,Global Biologics,700Chesterﬁeld Parkway West,Chesterﬁeld,Missouri63017Received14March2006;revised17May2006;accepted4June2006Published online in Wiley InterScience().DOI10.1002/jps.20727 ABSTRACT:The number of therapeutic monoclonal antibody in development hasincreased tremendously over the last several years and this trend continues.At presentthere are more than23approved antibodies on the US market and an estimated200ormore are in development.Although antibodies share certain structural similarities,development of commercially viable antibody pharmaceuticals has not been straightfor-ward because of their unique and somewhat unpredictable solution behavior.This articlereviews the structure and function of antibodies and the mechanisms of physical andchemical instabilities.Various aspects of formulation development have been examinedto identify the critical attributes for the stabilization of antibodies.ß2006Wiley-Liss,Inc.and the American Pharmacists Association J Pharm Sci96:1–26,2007Keywords:biotechnology;stabilization;protein formulation;protein aggregation;freeze drying/lyophilizationINTRODUCTIONProtein therapies are entering a new era with the inﬂux of a signiﬁcant number of antibody pharmaceuticals.Generally,protein drugs are effective at low concentrations with less side effects relative to small molecule drugs,even though,in rare cases,protein-induced antibody formation could be serious.1Therefore,this category of therapeutics is gaining tremendous momentum and widespread recognition both in small and large drugﬁrms.Among protein drug therapies,antibodies play a major role in control-ling many types of diseases such as cancer, infectious diseases,allergy,autoimmune dis-eases,and inﬂammation.Since the approval of theﬁrst monoclonal antibody(MAb)product -OKT-3in1986,more than23MAb drug products have entered the market(Tab.1).The estimated number of antibodies and antibody derivatives constitute20%of biopharmaceutical products currently in development(about200).2The global therapeutic antibody market was predicted to reach$16.7billion in2008.3There are several reasons for the increasing popularity of antibodies for commercial develop-ment.First,their action is speciﬁc,generally leading to fewer side effects.Second,antibodies may be conjugated to another therapeutic entity for efﬁcient delivery of this entity to a target site, thus reducing potential side effects.For instance, Mylotarg is an approved chemotherapy agent composed of calicheamicin conjugated to huma-nized IgG4,which binds speciﬁcally to CD33for the treatment of CD33-positive acute myeloid leukemia.Another example is the conjugation of immunotoxic barnase with the light chain of the anti-human ferritin monoclonal antibody F11as potential targeting agents for cancer immuno-therapy.4Third,antibodies may be conjugated to radioisotopes for speciﬁc diagnostic purposes. Examples include CEA-Scan for detection of color-ectal cancer and ProstaScint for detection of prostate stly,technology advancement has made complete human MAb available,which are lessimmunogenic.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY20071 Correspondence to:Wei Wang(Telephone:(636)-247-2111;Fax:(636)-247-5030;E-mail:wei.2.wang@pﬁ)Journal of Pharmaceutical Sciences,Vol.96,1–26(2007)Pharmacists AssociationT a b l e 1.C o m m e r c i a l M o n o c l o n a l A n t i b o d y P r o d u c t s#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H1A v a s t i n B e v a c i z u m a bH u m a n i z e d I g G 1,149k D a2004G e n e t e c h a n d B i o O n c o l o g y I V i n f u s i o nM e t a s t a t i c c a r c i n o m a o f c o l o n o r r e c t u m ,b i n d s V E G F 100m g a n d 400m g /v i a l (25m g /m L )s o l u t i o n 5.8m g /m L m o n o b a s i c N a P h o s H 2O ;1.2m g /m L d i b a s i c N a P h o s a n h y d r o u s (4m L ,16m L ﬁl l i n v i a l )60m g /m L a -T r e h a l o s e d i h y d r a t e (4m L ,16m L ﬁl l i n v i a l )0.4m g /m L P S 20(4m L ,16m L ﬁl l i n v i a l )6.22B e x x a rT o s i t u m o m a b a n d I -131T o s i t u m a b M u r i n e I g G 2l2003C o r i x a a n d G S KI V I n f u s i o nC D 20p o s i t i v e f o l l i c u l a r n o n H o d g k i n s l y m p h o m aK i t :14m g /m L M A b s o l u t i o n i n 35m g a n d 225m g v i a l s ;1.1m g /m L I 131-M A b s o l u t i o n10m M p h o s p h a t e (M A b v i a l )145m M N a C l ,10%w /v M a l t o s e ;I 131-M A b :5–6%P o v i d o n e ,1–2,9–15m g /m L M a l t o s e ,0.9m g /m L N a C l ,0.9–1.3m g /m L A s c o r b i c a c i d 7.23C a m p a t h A l e m t u z u m a bH u m a n i z e d ,I g G 1k ,150k D a2001I l e x O n c o l o g y ;M i l l e n i u m a n d B e r l e xI V i n f u s i o nB -c e l l c h r o n i c l y m p h o c y t i c l e u k e m i a ,CD 52-a n t i g e n 30m g /3m L s o l u t i o n3.5m g /3m L d i b a s i c N a P h o s ,0.6m g /3m L m o n o b a s i c K P h o s 24m g /3m L N a C l ,0.6m g /3m L K C l ,0.056m g /3m L N a 2E D T A 0.3m g /3m L P S 806.8–7.44C E A -S c a n (l y o )A c r i t u o m a b ;T c -99M u r i n e F a b ,50k D a1996I m m u n o m e d i c s I V i n j e c t i o n o r i n f u s i o nI m a g i n g a g e n t f o r c o l o r e c t a l c a n c e r1.25m g /v i a l L y o p h i l i z e d M A b .R e c o n s t i t u t e w 1m L S a l i n e w T c 99m 0.29m g /v i a l S t a n n o u s c h l o r i d e ,p o t a s s i u m s o d i u m t a r t r a t e t e t r a h y d r a t e ,N a A c e t a t e .3H 2O ,N a C l ,g l a c i a l a c e t i c a c i d ,H C l S u c r o s e5.75E r b i t u x C e t u x i m a bC h i m e r i c h u m a n /m o u s e I g G 1k ,152kD a 2004I m C l o n e a n d B M S I V i n f u s i o n T r e a t m e n t o fE GF R -e x p r e s s i n g c o l o r e c t a l c a r c i n o m a 100m g M A b i n 50m L ;2m g /m L s o l u t i o n1.88m g /m L D i b a s i c N a P h o s Á7H 2O ;0.42m g /m L M o n o b a s i c N a P h o s ÁH 2O8.48m g /m L N a C l 7.0–7.46H e r c e p t i n (l y o )T r a s t u z u m a bH u m a n i z e d I g G 1k1998G e n e t e c h I V i n f u s i o n M e t a s t a t i c b r e a s t c a n c e r w h o s e t u m o r o v e r e x p r e s s H E R 2p r o t e i n 440m g /v i a l ,21m g /m L a f t e r r e c o n s t i t u t i o n 9.9m g /20m L L -H i s t i d i n e H C l ,6.4m g /20m L L -H i s t i d i n e400m g /20m L a -T r e h a l o s e D i h y d r a t e 1.8m g /20m L P S 2067H u m i r a A d a l i m u m a bH u m a n I g G 1k ,148k D a2002C A T a n d A b b o t t S CR A p a t i e n t s n o t r e s p o n d i n g t o D M A R D s .B l o c k s T N F -a l p h a40m g /0.8m L s o l u t i o n (50m g /m L )0.69m g /0.8m L M o n o b a s i c N a P h o s Á2H 2O ;1.22m g /0.8m L D i b a s i c N a P h o s Á2H 2O ;0.24m g /0.8m L N a C i t r a t e ,1.04m g /0.8m L C i t r i c a c i d ÁH 2O 4.93m g /0.8m L N a C l ;9.6m g /0.8m L M a n n n i t o l 0.8m g /0.8m L P S 805.28L u c e n t i s R a n i b i z u m a bH u m a n i z e d I g G 1k f r a g m e n t2006G e n e n t e c h I n t r a v i t r e a l i n j e c t i o n A g e -r e l a t e d m a c u l a r d e g e n e r a t i o n (w e t )10m g /m L s o l u t i o n10m M H i s t i d i n e H C l10%a -T r e h a l o s e -D i h y d r a t e 0.01%P S 205.52WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jps9M y l o t a r g (l y o )G e m t u z u m a b o z o g a m i c i nH u m a n i z e d I g G 4k c o n j u g a t e d w i t h c a l i c h e a m i c i n2000C e l l t e c h a n d W y e t h I V i n f u s i o nH u m a n i z e d A b l i n k e d t o c a l i c h e a m i c i n f o r t r e a t m e n t o f C D 33p o s i t i v e a c u t e m y e l o i d l e u k e m i a 5m g p r o t e i n -e q u i v a l e n t l y o p h i l i z e d p o w d e r /20-m L v i a l M o n o b a s i c a n d d i b a s i c N a P h o s p h a t e D e x t r a n 40,S u c r o s e ,N a C l 10O n c o S c i n tS a t u m o m a b p e n d e t i d eM u r i n e I g G 1k c o n j u g a t e d t o G Y K -D T P A1992C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r c o l o r e c t a l a n d o v a r i a n c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (2m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 6.011O r t h o c l o n e O K TM u r o m o m a b -C D 3M u r i n e ,I g G 2a ,170k D a1986O r t h o B i o t e c h I V i n j e c t i o nR e v e r s a l o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n (a n t i C D 3-a n t i g e n )1m g /m L s o l u t i o n2.25m g /5m L m o n o b a s i c N a P h o s ,9.0m g /5m L d i b a s i c N a P h o s 43m g /5m L N a C l 1m g /m L P S 807Æ0.512P r o s t a S c i n tI n d i u m -111c a p r o m a b p e n d e t i d e M u r i n e I g G 1k -c o n j u g a t e d t o G Y K -D T P A1996C y t o g e n I V i n j e c t i o nI m a g i n g a g e n t f o r p r o s t a t e c a n c e r0.5m g c o n j u g a t e /m L s o l u t i o n (1m L p e r v i a l )P h o s p h a t e b u f f e r s a l i n e 5–713R a p t i v a (l y o )E f a l i z u m a bH u m a n i z e d I g G 1k2003X o m a a n d G e n e n t e c h S C C h r o n i c m o d e r a t e t o s e v e r e p l a q u e p s o r i a s i s ,b i n d s t o C D 11a s u b u n i t o f L F A -1150m g M A b /v i a l ;125m g /1.25m L (100m g /m L )a f t e r r e c o n s t i t u t i o n w i t h 1.3m L S W F I 6.8m g /v i a l L -H i s t i d i n e H C l ÁH 2O ;4.3m g /v i a l L -H i s t i d i n e123.2m g /v i a l S u c r o s e 3m g /v i a l P S 206.214R e m i c a d e (l y o )I n ﬂi x i m a bC h i m e r i c h u m a n /m u r i n e M A b a g a i n s t T N F a l p h a (a p p .30%m u r i n e ,70%c o r r e s p o n d s t o h u m a n I g G 1h e a v y c h a i n a n d h u m a n k a p p a l i g h t c h a i n c o n s t a n t r e g i o n s )1998C e n t o c o r I V i n f u s i o nR A a n d C r o h n ’s d i s e a s e (a n t i T N F a l p h a )100m g /20-m L V i a l ,10m g /m L o n r e c o n s t i t u t i o n2.2m g /10m L M o n o b a s i c N a P h o s H 2O ,6.1m g /10m L D i b a s i c N a P h o s Á2H 2O 500m g /10m L S u c r o s e 0.5m g /10m L P S 807.215R e o P r o A b c i x i m a bF a b .C h i m e r i c h u m a n -m u r i n e ,48k D a 1994C e n t o c o r /L i l l y I V i n j e c t i o n a n d i n f u s i o n R e d u c t i o n o f a c u t e b l o o d c l o t r e l a t e d c o m p l i c a t i o n s 2m g /m L s o l u t i o n 0.01M N a P h o s p h a t e 0.15M N a C l 0.001%(0.01m g /m L )P S 807.216R i t u x a n R i t u x i m a bC h i m e r i c m o u s e /h u m a n I g G 1k w i t h m u r i n e l i g h t a n d h e a v y c h a i n v a r i a b l e r e g i o n (F a b d o m a i n ),145kD a1997I D E C a n d G e n e n t e c h I V i n f u s i o nN o n H o d g k i n ’s l y m p h o m a .(a n t i C D 20-a n t i g e n )10m g /m L s o l u t i o n7.35m g /m L N a C i t r a t e Á2H 2O9m g /m L N a C l 0.7m g /m L P S 806.5(C o n t i n u e d )ANTIBODY FORMULATION3DOI 10.1002/jpsJOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 200717S i m u l e c t (l y o )B a s i l i x i m a bC h i m a r i c I g G 1k ,144kD a1998N o v a r t i s I V i n j e c t i o n a n d i n f u s i o nP r e v e n t i o n o f a c u t e k i d n e y t r a n s p l a n t r e j e c t i o n ,I L -2r e c e p t o r a n t a g o n i s t10m g a n d 20m g /v i a l ,4m g /m L o n r e c o n s t i t u t i o n 3.61m g ,7.21m g M o n o b a s i c K P h o s ;0.50m g ,0.99m g N a 2H P O 40.8m g ,1.61m g N a C l ;10m g ,20m g S u c r o s e ;40m g ,80m g M a n n i t o l ;20m g 40m g G l y c i n e 18S y n a g i s (l y o )P a l i v i z u m a bH u m a n i z e d I g G 1k ,C D R o f m u r i n e M A b 1129,148k D a 1998M e d I m m u n e I M i n j e c t i o nP r e v e n t r e p l i c a t i o n o f t h e R e s p i r a t o r y s y n c y t i a l v i r u s (R S V )50m g a n d 100m g /v i a l ,100m g /m L o n r e c o n s t i t u t i o n47m M H i s t i d i n e ,3.0m M G l y c i n e 5.6%M a n n i t o l19T y s a b r i N a t a l i z u m a bH u m a i n z e d I g G 4k2004B i o g e n I D E C I V I n f u s i o nM S r e l a p s e 300m g /15m L s o l u t i o n 17.0m g M o n o b a s i c N a P h o s ÁH 2O ,7.24m g d i B a s i c N a P h o s Á7H 2O f o r 15m L 123m g /15m L N a C l3.0m g /15m L P S 806.120V e r l u m a N o f e t u m o m a b M u r i n e F a b 1996B o e h r i n g e r I n g e l h e i m a n d D u P o n t M e r c k I V i n j e c t i o n I m a g i n g a g e n t f o r l u n g c a n c e r10m g /m L s o l u t i o nP h o s p h a t e b u f f e r s a l i n e?21X o l a i r (l y o )O m a l i z u m a bH u m a n i z e d I g G 1k ,149k D aG e n e n t e c h w N o v a r t i s a n d T a n o xS CA s t h m a ,i n h i b i t s b i n d i n g o f I g E t o I g E r e c e p t o r F C e R I202.5m g /v i a l ,D e l i v e r 150m g /1.2m L o n r e c o n s t i t u t i o n w i t h 1.4m L S W F I 2.8m g L H i s t i d i n e H C l ÁH 2O ;1.8m g L H i s t i d i n e145.5m g S u c r o s e 0.5m g P S 2022Z e n a p a x D a c l i z u m a bH u m a n i z e d I g G 1,144k D a1997R o c h e I V i n f u s i o nP r o p h y l a x i s o f a c u t e o r g a n r e j e c t i o n i n p a t i e n t s r e c e i v i n g r e n a l t r a n s p l a n t s .I n h i b i t s I L -2b i n d i n g t o t h e T a c s u b u n i t o f I L -2r e c e p t o r c o m p l e x 25m g /5m L M A b S o l u t i o n3.6m g /m L M o n o b a s i c N a P h o s ÁH 2O ;11m g /m L D i b a s i c N a P h o s Á7H 2O4.6m g /m L N a C l 0.2m g /m L P S 806.923Z e v a l i nI b r i t u m o m a b -T i u x e t a nM u r i n e I g G 1k -t h i o u r e a c o v a l e n t l i n k a g e t o T i u x e t a nI D E C I V i n f u s i o nC D 20a n t i g e n .(K i t w i t h Y t t e r i u m -90i n d u c e s c e l l u l a r d a m a g e b y b e t a e m i s s i o n )3.2m g /2m L s o l u t i o n 09%N a C l 7.1T a b l e 1.(C o n t i n u e d )#B r a n d n a m e M o l e c u l eM A bY e a r C o m p a n y R o u t e I n d i c a t i o n M A b C o n c B u f f e r E x c i p i e n t s S u r f a c t a n t p H4WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY 2007DOI 10.1002/jpsDevelopment of commercially viable antibody pharmaceuticals has,however,not been straight-forward.This is because the behavior of antibodies seems to vary,even though they have similar structures.In attempting to address some of the challenges in developing antibody therapeutics, Harris et al.5reviewed the commercial-scale formulation and characterization of therapeutic recombinant antibodies.In a different review, antibody production and puriﬁcation have been discussed.2Nevertheless,the overall instability and stabilization of antibody drug candidates have not been carefully examined in the litera-ture.This article,not meant to be exhaustive, intends to review the structure and functions of antibodies,discuss their instabilities,and sum-marize the methods for stabilizing/formulating antibodies.ANTIBODY STRUCTUREAntibodies(immunoglobulins)are roughly Y-shaped molecules or combination of such molecules(Fig.1). Their structures are divided into two regions—the variable(V)region(top of the Y)deﬁning antigen-binding properties and the constant(C)region (stem of the Y),interacting with effector cells and molecules.Immunoglobulins can be divided into ﬁve different classesÀIgA,IgD,IgE,IgM,and IgG based on their C regions,respectively desig-nated as a,d,e,m,and g(ﬁve main heavy-chain classes).6Most IgGs are monomers,but IgA and IgM are respectively,dimmers and pentamers linked by J chains.IgGs are the most abundant,widely used for therapeutic purposes,and their structures will be discussed as antibody examples in detail.Primary StructureThe structure of IgGs have been thoroughly reviewed.6The features of the primary structure of antibodies include heavy and light chains, glycosylation,disulﬁde bond,and heterogeneity. Heavy and Light ChainsIgGs contain two identical heavy(H,50kDa)and two identical light(L,25kDa)chains(Fig.1). Therefore,the total molecular weight is approxi-mately150kDa.There are several disulﬁde bonds linking the two heavy chains,linking the heavy and light chains,and residing inside the chains (also see next section).IgGs are further divided into several subclasses—IgG1,IgG2,IgG3,and IgG4(in order of relative abundance in human plasma),with different heavy chains,named g1, g2,g3,and g4,respectively.The structural differences among these subtypes are the number and location of interchain disulﬁde bonds and the length of the hinge region.The light chains consist of two types—lambda(l)and kappa(k). In mice,the average of k to l ratio is20:1,whereas it is2:1in humans.6The variable(V)regions of both chains cover approximately theﬁrst 110amino acids,forming the antigen-binding (Fab)regions,whereas the remaining sequences are constant(C)regions,forming Fc(fragment crystallizable)regions for effector recognition and binding.6The N-terminal sequences of both the heavy and light chains vary greatly between different antibodies.It was suggested that the conserved sequences in human IgG1antibodies Figure1.Linear(upper panel)and steric(lower panel)structures of immunoglobulins(IgG).ANTIBODY FORMULATION5DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007are approximately95%and the remaining5% is variable and creates their antigen-binding speciﬁcity.5The V regions are further divided into three hypervariable sequences(HV1,HV2,and HV3)on both H and L chains.In the light chains,these are roughly from residues28to35,from49to59,and from92to103,respectively.6Other regions are the framework regions(FR1,FR2,FR3,and FR4).The HV regions are also called the complementarity determining regions(CDR1,CDR2,and CDR3). While the framework regions form the b-sheets, the HV sequences form three loops at the outer edge of the b barrel(also see Section2.2).Disulﬁde BondsMost IgGs have four interchain disulﬁde bonds—two connecting the two H chains at the hinge region and the other two connecting the two L chains to the H chains.6Exceptions do exist.Two disulﬁde bonds were found in IgG1and IgG4 linking the two heavy chain in the hinge region but four in IgG2.7In IgG1MAb,HC is linked to the LC between theﬁfth Cys(C217)of HC and C213on the LC.In IgG2and IgG4MAbs,it is the third Cys of HC(C123)linking to the LC.7A disulﬁde bond between HC C128and LC C214 was found for mouse catalytic monoclonal anti-bodies(IgG2a).8IgGs have four intrachain disulﬁde bonds, residing in each domain of the H and L chains, stabilizing these domains.The intrachain disul-ﬁde bonds in V H and V L are required in functional antigen binding.9Native IgG MAbs should not have any free sulfhydryl groups.7However, detailed examination of the free sulfhydryl groups in recombinant MAbs(one IgG1,two IgG2,and one IgG4)suggests presence of a small portion of free sulfhydryl group(approximately0.02mol per mole of IgG2or IgG4MAb and0.03for IgG1.7In rare cases,a free cysteine is found.A nondisulﬁde-bonded Cys at residue105was found on the heavy chain of a mouse monoclonal antibody,OKT3 (IgG2a).10OligosaccharidesThere is one oligosaccharide chain in IgGs.6This N-linked biantennary sugar chain resides mostly on the conserved Asn297,which is buried between the C H2domains.5,11For example,the oligosaccharide resides on Asn-297of the C H2 domain of chimeric IgG1and IgG3molecules12but on Asn299in a monoclonal antibody,OKT3 (IgG2a).10The oligosaccharide,often microheter-ogeneous,is typically fucosylated in antibodies produced in CHO or myeloma cell lines5and may differ in other cell lines.2,11There are many factors that dictate the nature of the glycan microheterogenity on IgGs.These include cell line,the bioreactor conditions and the nature of the downstream processing.An additional oligo-saccharide can be found in rare cases.A human IgG produced by a human-human-mouse hetero-hybridoma contains an additional oligosaccharide on Asn75in the variable region of its heavy chain.13In addition,O-linked carbohydrates could also exist in this antibody.Proper glycosylation is critical for correct functioning of antibodies.11It was demonstrated that removal of the oligosaccharide in IgGs(IgG1 and IgG3)made them ineffective in binding to C1q, in binding to the human Fc g RI and activating C; and generally more sensitive to most proteases than their corresponding wild-type IgGs(one exception).12This is because the binding site on IgG for C1q,theﬁrst component of the complement cascade,is localized in the C H2domains.11 Furthermore,the glycosylation can affect the antibody conformation.12Oligosaccharides in other regions can also play a critical role.Removal of an oligosaccharide in a Fv region of the CBGA1antibody resulted in a decreased antigen-binding activity in several ELISA systems.13In addition,this oligosaccharide might play critical role in reducing the antigenicity of the protein.14The sugar composition of the oligosaccharide is also critical in antibody functions.It has been shown that a low fucose(Fuc)content in the complex-type oligosaccharide in a humanized chimeric IgG1is responsible for a50-fold higher antibody-dependent cellular cytotoxicity(ADCC) compared with a high Fuc counterpart.15 HeterogeneityPuriﬁed antibodies are heterogeneous in struc-ture.This is true for all monoclonal antibodies (MAbs)due to differences in glycosylation pat-terns,instability during production,and terminal processing.5For example,ﬁve charged isoforms were found in recombinant humanized monoclo-nal antibody HER2as found by capillary iso-electric focusing(cIEF)and sodium dodecyl sulfate–capillary gel electrophoresis(SDS–CGE).16Six separate bands were focused under6WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jpsIEF for two mouse monoclonal antibodies IgG2a (k)and IgG1(k).17A mature monoclonal antibody, OKT3(IgG2a),contain cyclized N-terminus (pyroglutamic acid,À17D)in both H and L chains, processed C-terminus(no Lys,À128D)of the H chains,and a small amount of deamidated form.10 Similar observation was also reported for a huma-nized IgG1(k).18In rare cases,gene cross-over may lead to formation of abnormal heavy chains.For example,a puriﬁed monoclonal anti-IgE antibody contains a small amount of a variant H chain, which had16fewer amino acid residues than the normal H chain(position is between Arg108of the L chain and Ala124of the H chain).19 Secondary and Higher-Order StructureThe basic secondary and higher-order structural features of IgGs have been reviewed.6Only a small portion of the three-dimensional structures of IgGs has been solved.20The antibody’s secon-day structure is formed as the polypeptide chains form anti-parallel b-sheets.The major type of secondary structure in IgGs is these b-sheets and its content is roughly70%as measured by FTIR.21The light chain consists of two and the heavy chain contains four domains,each about 110amino acid long.6,20All these domains have similar folded structures—b barrel,also called immunoglobulin fold,which is stabilized by a disulﬁde bond and hydrophobic interaction(pri-mary).These individual domains($12kDa in size)interact with one another(V H and V L;C H1 and C L;and between two C H3domains except the carbohydrate-containing C H2domain)and fold into three equal-sized spherical shape linked by a ﬂexible hinge region.These three spheres form a Y shape(mostly)and/or a T shape.22The less globular shape of IgGs is maintained both by disulﬁde bonds and by strong noncovalent interactions between the two heavy chains and between each of the heavy-chain/light-chain pairs.23Through noncovalent interactions,a less stable domain becomes more stable,and thus,the whole molecule can be stabilized.24A detailed study indicates that the interaction between two CH3domains are dominated by six contact residues,ﬁve of these residues(T366,L368, F405,Y407,and K409)forming a patch at the center of the interface.25These noncovalent interactions are spatially oriented such that variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)induces noncovalent multimerization.26The six hypervariable regions in CDR(L1,L2, L3,H1,H2,and H3)form loops of a few predictable main-chain conformations(or canonical forms), except H3loop,which has too many variations in conformation to be predicted accurately.27,28 There is a slight difference in the loop composition and shape between the two types of light chains.20 However,no functional difference was found in antibodies having l or k chain.6Basic Functions of AntibodiesThe basic functions of antibodies have been reviewed.6There are two functional areas in IgGs—the V and C regions.The V regions of the two heavy and light chains offer two identical antigen-binding sites.The binding of the two sites (bivalent)can be independent of each other and does not seem to depend on the C region.29The exact antigen-binding sites are the CDR regions with participation of the frame work regions.30 Binding of antigens seems through the induced-ﬁt mechanism.31,32The induced-ﬁt mechanism allows multispeciﬁcity and polyreactivity.It has been suggested that about5–10residues usually contribute signiﬁcantly to the binding energy.32 The C regions of antibodies have three main effector functions(1)being recognized by receptors on immune effector cells,initiating antibody-dependent cell cytotoxicities(ADCC),(2)binding to complement,helping to recruit activated pha-gocytes,and(3)being transported to a variety of places,such as tears and milk.6In addition,C domains also modulate in vivo stability.23,29,33The function of Fc is affected by the structure of Fab. Variable domain exchange(switching V H and V L; inside-out IgG;ioIgG)affected Fc-associated func-tions such as serum half-life and binding to protein G and Fc g RI.26The hinge region providesﬂexibility in bivalent antigen binding and activation of Fc effector functions.26Two chimeric IgG3antibodies lacking a genetic hinge but with Cys residues in CH2 regions was found to be deﬁcient in their inter-molecular assembly,and both IgG3D HþCys and IgG3D Hþ2Cys lost greatly their ability to bind Fc g RI and failed to bind C1q and activate the complement cascade.34Alternative Forms of AntibodiesIn addition to species-speciﬁc antibodies,other antibody forms are generated to meet various needs.In the early development of antibody therapies,antibodies were made from murineANTIBODY FORMULATION7DOI10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007sources.However,these antibodies easily elicit formation of human anti-mouse antibody (HAMA).Therefore,humanized chimeric antibo-dies were generated.Chimeric monoclonal anti-bodies(60–70%human)are made of mouse variable regions and human constant regions.2 Such antibodies can still induce formation of human anti-chimeric antibody(HACA).Highly humanized antibodies,CDR-grafted antibodies, are made by replacing only the human CDR with mouse CDR regions(90–95%human).2These antibodies are almost the same in immunogeni-city potential as completely human antibodies, which may illicit formation of human anti-human antibody(HAHA).Other alternative forms of antibodies have also been generated and these different forms have been reviewed.35Treatment with papain would cleave the N-terminal side of the disulﬁde bonds and generate two identical Fab fragments and one Fc fragment.Fab0s are50kDa(V HþC H1)/ (V LþC L)heterodimers linked by a single disul-ﬁde bond.Treatment with pepsin cleaves the C-terminal side of the disulﬁde bonds and pro-duces a F(ab)02fragment.The remaining H chains were cut into several small fragments.6Cleavage by papain occurs at the C-terminal side of His-H22836or His-H227.37Reduction of F(ab0)2will produce two Fab0.23Fv fragments are noncovalent heterodimers of V H and V L.Stabilization of the fragment by a hydrophilicﬂexible peptide linker generates single-chain Fv(scFvs).2Fragments without constant domains can also be made into domain antibodies (dAbs).These scFvs are25–30kDa variable domain (V HþV L)dimers joined by polypeptide linkers of at least12residues.Shorter linkers(5–10residues)do not allow pairing of the variable domains but allow association with another scFv form a bivalent dimer (diabody)(about60kDa,or trimer:triabody about 90kDa).38Two diabodies can be further linked together to generate bispeciﬁc tandem diabody (tandab).39Disulﬁde-free scFv molecules are rela-tively stable and useful for intracellular applica-tions of antibodies—‘‘intrabodies.’’38The smallest of the antibody fragments is the minimal recognition unit(MRU)that can be derived from the peptide sequences of a single CDR.2ANTIBODY INSTABILITYAntibodies,like other proteins,are prone to a variety of physical and chemical degradation path-ways,although antibodies,on the average,seem to be more stable than other proteins.Antibody instabilities can be observed in liquid,frozen,and lyophilized states.The glycosylation state of an antibody can signiﬁcantly affect its degradation rate.40In many cases,multiple degradation path-ways can occur at the same time and the degrada-tion mechanism may change depending on the stress conditions.41These degradation pathways are divided into two major categories—physical and chemical instabilities.This section will explore the possible degradation pathways of antibodies and their inﬂuencing factors.Physical InstabilityAntibodies can show physical instability via two major pathways—denaturation and aggregation. DenaturationAntibodies can denature under a variety of conditions.These conditions include temperature change,shear,and various processing steps. Compared with other proteins,antibodies seem to be more resistant to thermal stress.They may not melt completely until temperature is raised above708C,21,42,43while most other mesophilic proteins seem to melt below708C.44Shear may cause antibody denaturation.For example,the antigen-binding activity of a recombinant scFv antibody fragment was reduced with aﬁrst-order rate constant of0.83/h in a buffer solution at a shear of approximately20,000/s.45Lyophilization can denature a protein to var-ious extents.An anti-idiotypic antibody(MMA 383)in a formulation containing mannitol,sac-charose,NaCl,and phosphate was found to loose its in vivo immunogenic properties(only10–20% of normal response rate)upon lyophilization.46 Since the protein showed no evidence of degrada-tion after lyophilization,no change in secondary structure by CD(29%b-sheet,14%a-helix,and 57%‘‘other’’),the loss of activity was attributed to the conformational change.Indeed,tryptophan ﬂuorescence properties were different between the lyophilized and unlyophilized antibodies.46 AggregationAntibody aggregation is a more common manifes-tation of physical instability.The concentration-dependent antibody aggregation was considered the greatest challenge to developing protein formulations at higher concentrations.47This is8WANG ET AL.JOURNAL OF PHARMACEUTICAL SCIENCES,VOL.96,NO.1,JANUARY2007DOI10.1002/jps。

Phanta超高保真酶

引物设计注意事项
1.引物3’端最后一个碱基选择C或G； 2.引物3’端最后8个碱基应避免出现连续错配； 3.引物3’端尽量避免出现发夹结构； 4.引物Tm值控制在55℃-65℃之间； 5.引物额外附加序列，即与模板非配对序列，不应参与引物Tm值计算； 6.引物GC含量控制在40%-60%之间； 7.正向引物和反向引物Tm值以及GC含量尽可能一致。
应用实例
1.反应体系配制：
所有操作请在冰上进行，各组分解冻后请充分摇匀。为了防止PhantaTM Super-Fidelity DNA Polymerase的校对活性降解引物，请将聚合酶最后加入反应体系中。各组分使用完毕后及时放回-20℃。5 × SF Buffer请勿长时间敞口放置。
ddH2O 5 × SF Buffer (with 10 mM MgSO4) 25 mM MgSO4a dNTP Mix (10 mM each)b
a. 对于大多数PCR反应，Mg2+最佳终浓度为1.5-2 mM。体系中已含有终浓度为2 mM Mg2+，如有需要，可用25 mM MgSO4，以0.2-0.5 mM为间隔向上摸索Mg2+最佳使用浓度。
b. 请勿使用dUTP和带有尿嘧啶的引物或模板。 c. 扩增子GC含量>60%时加入终浓度3%的DMSO有可能会有助于扩增。 d. 推荐仅当扩增子GC含量>60%且优化条件也无法正常扩增时使用；可能会降低保真度。 e. 不同模板最佳反应浓度有所不同，下表为50 μl反应体系推荐模板使用量：
DMSOc
5 × PCR Enhancerd
模板DNAe
引物1 (10 μM)
引物2 (10 μM)
PhantaTM Super-Fidelity DNA Polymerase (1 U/μl)f

日本大白兔PRLR基因外显子10的克隆与测序

日本大白兔PRLR基因外显子10的克隆与测序杨洪雁;胡锐;王星;白秀娟【摘要】采用聚合酶链式反应方法,从日本大白兔血液中提取DNA,扩增出泌乳素受体基因(PRLR)外显子10,并对其进行克隆与测序,获得长度分别为208 bp和224 bp的2段序列,经过拼接得到1段418 bp的序列.将序列提交到GenBank上,GenBank中的Blast分析表明,测序得到的日本大白兔PRLR基因与家兔的同源性为99%.通过与其他目动物比较,PRLR基因在不同目动物中同源性不高,而在同目动物中同源性很高,表明PRLR基因在同一目中具有较高的保守性,同时在进化过程中具有一定的物种特异性.【期刊名称】《江苏农业科学》【年(卷),期】2010(000)001【总页数】3页(P59-61)【关键词】PRLR基因;克隆;测序;日本大白兔【作者】杨洪雁;胡锐;王星;白秀娟【作者单位】东北农业大学动物科学技术学院,黑龙江哈尔滨,150030;辽东学院农学院,辽宁丹东118003;东北农业大学动物科学技术学院,黑龙江哈尔滨,150030;辽东学院农学院,辽宁丹东118003;东北农业大学动物科学技术学院,黑龙江哈尔滨,150030【正文语种】中文【中图分类】S813催乳素(prolactin,PRL)又称促乳素或生乳素，是主要由垂体前叶催乳素细胞合成和分泌的一种蛋白质激素，是含199个氨基酸并有3个二硫键的多肽，相对分子质量为22000，主要作用是促进乳腺发育生长，引起并维持泌乳，是动物繁殖成功必需的激素之一。

催乳素要行使其功能，必须通过位于靶细胞膜上的催乳素受体(prolactin receptor,PRLR)介导才能完成，PRLR基因的翻译产物为含1个跨膜区域的跨膜蛋白，由膜外域、跨膜域和胞内域3部分组成，具有细胞因子受体超家族的结构特征[1]。

哺乳动物中至少存在长型受体(long form)和短型受体(short form)2种类型[2]。

益生菌对阿尔茨海默病作用的研究进展

益生菌对阿尔茨海默病作用的研究进展发布时间：2021-12-14T06:08:15.523Z 来源：《中国结合医学杂志》2021年12期作者：宋鑫萍1,2，李盛钰2，金清1[导读] 阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

近年来研究显示，益生菌在适量使用时作为有益于宿主健康的微生物，在防治阿尔茨海默病方面具有积极影响，其作用机制可能通过调节肠道菌群，影响神经免疫系统，调控神经活性物质以及代谢产物，通过肠-脑轴影响该病发生和发展。

宋鑫萍1,2，李盛钰2，金清11.延边大学农学院，吉林延吉 1330022.吉林省农业科学院农产品加工研究所，吉林长春 130033摘要：阿尔茨海默病已成为威胁全球老年人生命健康的主要疾病之一，患者数量逐年攀升，其护理的经济成本高，给全球经济造成重大挑战。

本文综述了近几年来国内外益生菌对阿尔茨海默病的作用进展，以及其预防和治疗阿尔茨海默病的潜在作用机制。

关键词：益生菌；阿尔茨海默病；肠道菌群；机制Recent Progress in Research on Probiotics Effect on Alzheimer’s DiseaseSONG Xinping1,2，LI Shengyu2，JI Qing1*(1.College of Agricultural, Yanbian University, Yanji 133002，China)(2.Institute of Agro-food Technology, Jilin Academy of Agricultural Sciences, Chanchun 130033, China)Abstract：Alzheimer’s disease has become one of the major diseases threatening the life and health of the global elderly. The number of patients is increasing year by year, and the economic cost of nursing is high, which poses a major challenge to the global economy. In recent years, studies have shown that probiotics, as microorganisms beneficial to the health of the host, have a positive impact on the prevention and treatment of Alzheimer’s disease. Its mechanism may be through regulating intestinal flora, affecting the nervous immune system, regulating the neuroactive substances and metabolites, and affecting the occurrence and development of the disease through thegut- brain axis. This paper reviews the progress of probiotics on Alzheimer’s disease at home and abroad in recent years, as well as its potential mechanism of prevention and treatment.Key words：probiotics; Alzheimer’s disease; gut microbiota; mechanism阿尔茨海默病（Alzheimer’s disease, AD），系中枢神经系统退行性疾病，属于老年期痴呆常见类型，临床特征主要包括：记忆力减退、认知功能障碍、行为改变、焦虑和抑郁等。

拟南芥

The Conserved Splicing Factor SUA Controls Alternative Splicing of the Developmental Regulator ABI3in Arabidopsis W OAMatteo Sugliani,a Vittoria Brambilla,a Emile J.M.Clerkx,b,1Maarten Koornneef,a,b and Wim J.J.Soppe a,2a Department of Plant Breeding and Genetics,Max Planck Institute for Plant Breeding Research,50829Cologne,Germanyb Laboratory of Genetics,Wageningen University,6708PB Wageningen,The NetherlandsABSCISIC ACID INSENSITIVE3(ABI3)is a major regulator of seed maturation in Arabidopsis thaliana.We detected two ABI3 transcripts,ABI3-a and ABI3-b,which encode full-length and truncated proteins,respectively.Alternative splicing of ABI3is developmentally regulated,and the ABI3-b transcript accumulates at the end of seed maturation.The two ABI3transcripts differ by the presence of a cryptic intron in ABI3-a,which is spliced out in ABI3-b.The suppressor of abi3-5(sua)mutant consistently restores wild-type seed features in the frameshift mutant abi3-5but does not suppress other abi3mutant alleles.SUA is a conserved splicing factor,homologous to the human protein RBM5,and reduces splicing of the cryptic ABI3intron,leading to a decrease in ABI3-b transcript.In the abi3-5mutant,ABI3-b codes for a functional ABI3protein due to frameshift restoration.INTRODUCTIONSeeds are essential for the spread and survival of most plant species and constitute a major food source.Seed features like desiccation tolerance,dormancy,and the accumulation of stor-age proteins are established during seed maturation.In Arabi-dopsis thaliana,the phytohormone abscisic acid(ABA)controls seed maturation and dormancy by preventing germination and reserve mobilization.ABA signaling at this stage is concomitant with the expression of four major regulatory genes of seed maturation with partially redundant functions:LEAFY COTYLE-DON1(LEC1),LEC2,FUSCA3(FUS3),and ABSCISIC ACID INSENSITIVE3(ABI3)(Kroj et al.,2003;To et al.,2006).ABI3is a main component of the ABA signaling pathway and is highly conserved among plant species.The ABI3protein contains four functional domains(Giraudat et al.,1992;Suzuki et al.,1997). The A1domain is an acidic transcriptional activator(McCarty et al.,1991),and B1can interact with the seed-speciﬁc tran-scription factor ABI5(Nakamura et al.,2001).B2and B3are two basic DNA binding domains responsible for the ABA-dependent activation of seed maturation genes(Suzuki et al.,1997;Ezcurra et al.,2000;Nag et al.,2005).Several abi3mutant alleles were isolated in Arabidopsis.One of the most severe is abi3-5,which was originally identiﬁed by its stay-green seed phenotype.abi3-5seeds are insensitive to ABA during germination,are desiccation intolerant,and have reduced longevity,similar to other strong abi3alleles(Ooms et al.,1993).ABI3transcription is promoted by LEC1,LEC2,FUS3,ABI3(To et al.,2006),and ABA(Lopez-Molina et al.,2002).During germi-nation,ABI3is repressed by the chromatin remodeling factor PICKLE(Perruc et al.,2007)and the ABI3protein is targeted to 26S proteasome degradation by the ABI3-INTERACTING PRO-TEIN2(Zhang et al.,2005).The identiﬁcation of several splice variants of ABI3homologs in monocotyledon and dicotyledon species(McKibbin et al.,2002;Fan et al.,2007;Gagete et al., 2009)implies that alternative splicing also has an important role in controlling ABI3expression.However,splicing variants of ABI3were not observed in Arabidopsis.Although alternative splicing of mRNA is an important com-ponent of posttranscriptional regulation in higher eukaryotes,its relevance and mechanisms in plants are poorly understood.In Arabidopsis,;42%of all transcripts from intron-containing genes are alternatively spliced(Filichkin et al.,2010).Alternative splicing can produce transcripts that encode for proteins with altered or lost function.Furthermore,it can lead to tissue-speciﬁc transcripts or affect mRNA stability and turnover via nonsense-mediated decay(McGlincy and Smith,2008).Splicing is directed by the spliceosome,a dynamic RNA-protein multicomponent machinery that is conserved among eukaryotes.In Arabidopsis, only a few splicing-related proteins have been characterized (Lopato et al.,1999;Ali et al.,2007;Tanabe et al.,2007;Zhang and Mount,2009),and the information on their biochemical function and their targets in relevant developmental and envi-ronmental contexts is limited.We identiﬁed SUPPRESSOR OF ABI3-5(SUA)as a novel plant splicing factor that inﬂuences seed maturation by controlling alternative splicing of ABI3.SUA is an evolutionary conserved protein that suppresses splicing of a cryptic ABI3intron.Splicing of this intron leads to a transcriptThe authors responsible for distribution of materials integral to theﬁndings presented in this article in accordance with the policy describedin the Instructions for Authors()are:Matteo Sugliani(sugliani@mpipz.mpg.de)and Wim J.J.Soppe(soppe@mpipz.mpg.de).1Current address:University of Applied Sciences,Hogeschool HAS DenBosch,5223DE,Hertogenbosch,The Netherlands.2Address correspondence to soppe@mpipz.mpg.de.W Online version contains Web-only data.OA Open Access articles can be viewed online without a subscription./cgi/doi/10.1105/tpc.110.074674The Plant Cell,Vol.22:1936–1946,June2010,ã2010American Society of Plant Biologiststhat encodes a truncated ABI3protein in the wild type but a functional protein in the abi3-5mutant background. RESULTSIsolation of the abi3-5sua-1Double MutantSeeds of the abi3-5glabra1(gl1)transparent testa5-1(tt5-1)triple mutant were mutagenized by g-irradiation to isolate mutants involved in ABI3signaling.The gl1and tt5-1mutations are located on both sides of the ABI3locus and were used as phenotypic markers to distinguish suppressor mutants from wild-type contaminants.A strong suppressor mutant of abi3-5 was identiﬁed in the M2generation and named suppressor of abi3-5(sua-1).The gl1-1and tt5-1mutations were removed from this line by backcrossing with its wild-type Landsberg erecta (L er)genetic background and subsequent selection for abi3-5 and sua-1in the progeny.Ripe abi3-5seeds are green due to the presence of chlorophyll,but abi3-5sua-1seeds are yellow-brown,similar to the wild type(Figure1A).In addition,abi3-5 seeds are nondormant and sensitive to desiccation,which causes reduced longevity.Seeds of abi3-5sua-1are also non-dormant,but their longevity is strongly improved and they still germinate nearly100%after10weeks of storage(Figure1B). Finally,abi3-5sua-1seeds show an increased sensitivity to ABA and cannot germinate on15m M ABA,whereas viable abi3-5mutant seeds show100%germination on30m M ABA (Figure1C).Identiﬁcation of the SUA GeneInitial mapping indicated that the sua mutation is located on chromosome 3.Fine-mapping was performed using an F2 mapping population of;4000individuals derived from a cross between the abi3-5sua-1double mutant(in L er background)and Columbia(Col-0).The abi3-5sua-1double mutant was identiﬁed in this mapping population by its yellow-brown seed color trait in combination with the ability to germinate in the presence of5m m ABA.The location of the sua-1mutation was narrowed down to a region of64kb at the bottom of chromosome3between two markers located at20.056and20.120Mb.This region contains 17genes and did not show recombination in ourmappingFigure1.The sua Mutation Suppresses abi3-5Phenotypes.(A)Seeds of wild type(L er and Col-0),abi3-5(in L er background),abi3-5sua-1(in L er background),abi3-5sua-2(in L er/Col-0background)and abi3-5 sua-1PSUA:SUA:GFP#8(in L er background).(B)Germination of L er,abi3-5,and abi3-5sua-1seeds after different periods of dry storage.Harvested seeds were stored at208C and42%relative humidity.Percentages are means(6SE)of three biological replicates.(C)Germination of L er,abi3-5,abi3-5sua-1,abi3-5sua-2,and abi3-5sua-3seeds,imbibed at different ABA concentrations.Seeds were1week after-ripened and stratiﬁed4d.Percentages are means(6SE)of four biological replicates.(D)Germination of1-week-old abi3-5,abi3-5sua-1PSUA:SUA:GFP#8,and abi3-5sua-1PSUA:SUA:GFP#15seeds at different ABA concentrations. Percentages are means(6SE)of three biological replicates.(E)Germination of L er,sua-1,Col-0,and sua-2seeds,imbibed at different ABA concentrations.Seeds were6months after-ripened and4d stratiﬁed. Percentages are means(6SE)of four biological replicates.SUA Controls Alternative Splicing of ABI31937parison of sequenced candidate genes with sequences in The Arabidopsis Information Resource (Garcia-Hernandez et al.,2002)revealed a 47-bp deletion in the 15th exon of At3G54230in the abi3-5sua-1double mutant (Fig-ure 2A).The identity of At3G54230as the SUA gene was conﬁrmed by complementation of the sua-1mutant in the abi3-5background.A construct containing the SUA cDNA,expressed from a 2711-bp putative SUA promoter and fused with a C-terminal green ﬂuorescent protein (GFP )tag (PSUA:SUA:GFP ),was used to transform abi3-5sua-1plants.Two independent T2transform-ants,containing a single insertion event,both complemented sua-1and showed the abi3-5phenotype.One of these trans-formants,abi3-5sua-1PSUA:SUA:GFP #8,even showed an enhanced abi3-5phenotype,yielding seeds with a more intense green color and stronger ABA insensitivity (Figures 1A and 1D).Additional mutant alleles of SUA in the Col-0background (sua-2and sua-3)were obtained from the Salk insertion mutant collection and from the GABI-Kat collection.These lines contain T-DNA insertions in the fourth and the ninth intron and were named sua-2and sua-3,respectively (Figure 2A).Both alleles lack full-length SUA expression and were crossed with abi3-5.The double mutants abi3-5sua-2and abi3-5sua-3were selected in the resulting F2,and all of them showed suppression of the abi3-5phenotypes,similar to abi3-5sua-1(Figures 1A and 1C).The sua single mutants did not have any obvious visual phenotype.Detailed analysis revealed that sua-1seeds are more susceptible to ABA germination inhibition compared with wild-type L er .By contrast,sua-2seeds germinated better than wild-type Col-0in the presence of ABA (Figure 1E).SUA Encodes an RNA Binding Protein Located in the Nucleus and Expressed in All Plant TissuesSUA encodes a protein with a conserved domain architecture that suggests a function in RNA metabolism.SUA contains two RNA recognition motifs surrounding a Zinc ﬁnger domain,an octamer repeat domain,and a Gly-rich domain close to the carboxy end (Figure 2B).The Arabidopsis genome does not contain a second gene with this combination of domains.SUA homologs,however,can be found throughout the eukaryotic kingdom (Figure 2C).SUA has 45%sequence similarity with the human RNA Binding Motif Protein 5(RBM5),which was originally identiﬁed as a putative tumor suppressor gene that is part of a small gene family (Edamatsu et al.,2000).Publicly available microarray data (Zimmermann et al.,2004)show ubiquitous SUA expression in Arabidopsis ,with a moder-ate enrichment in seeds.Quantitative real-time RT-PCR analysis conﬁrmed that the relative abundance of SUA transcripts is comparable in most Arabidopsis tissues,but highest in siliques toward the end of seed maturation (Figure 3A).The subcellular localization of the SUA protein was studied using the PSUA:SUA:GFP lines.A GFP signal was detected in the nucleus of vegeta-tive and reproductive tissues (Figure 3B).The SUA_GFP chimeric protein showed diverse patterns.Speckles of different size were observed in some nuclei,but ﬂuorescence was diffuse and rather weak in others (Figure 3B).We did not observe a correlation between the SUA_GFP ﬂuorescence pattern and tissue or de-velopmental stages.SUA Interacts with the Prespliceosomal Component U2AF 65RBM5,the human homolog of SUA,is a member of the prespliceosomal complex (Behzadnia et al.,2007)and interacts with U2AF 65in vivo (Bonnal et al.,2008).U2AF 65is the larger subunit of the conserved pre-mRNA splicing factor U2AF.It guides splice site selection during the formation of the spliceo-somal complex (Zamore et al.,1992;Sickmier et al.,2006).In a yeast two-hybrid GAL4assay,we detected interaction between SUA and Arabidopsis U2AF 65(BAH19725)(Domon et al.,1998;Figure 4A).To conﬁrm the SUA-U2AF 65interaction in planta,we performed a ﬂuorescence resonance energy transfer/ﬂuo-rescence lifetime imaging (FRET/FLIM)assay.Arabidopsis leaf protoplasts were cotransfected with two vectors fortheFigure 2.Genetic Structure,Domain Organization,and Phylogenetic Relationships of SUA .(A)Schematic structure of the SUA gene.Triangles indicate the T-DNA insertion sites of sua-2and sua-3,and the dashed region represents the 47-bp deletion of the sua-1allele.UTRs are shown in white,exons in gray,and introns as thick lines.(B)Domain structure of the SUA protein.aa,amino acids;RRM,RNA recognition motif;Zn,zinc ﬁnger;OCRE,octamer repeat;G-p,Gly patch.(C)Phylogram of SUA and its closest related proteins.FCA is an RNA binding protein that was added to the tree to emphasize the similarity between SUA and its homologs in evolutionary distant species.Populus trichocarpa (Pt ),Vitis vinifera (Vv ),Oryza sativa (Os ),Physcomitrella patens (Pp ),Chlamydomonas reinhardtii (Cr ),Xenopus laevis (Xl ),Mus musculus (Mm ),and Homo sapiens (Hs ).Bootstrap values are shown when higher than 50.1938The Plant Celloverexpression of SUA_YFP (yellow ﬂuorescent protein)and U2AF 65_CFP chimerical proteins.FRET/FLIM analysis of proto-plasts coexpressing SUA_YFP and U2AF 65_CFP (cyan ﬂuores-cent protein)showed a signiﬁcant reduction of the mean CFP ﬂuorescence lifetime compared with those expressing the U2AF 65-CFP alone (Figures 4B to 4F),conﬁrming interaction of both proteins in planta.The Suppression of abi3-5by sua-1Is Allele Speciﬁc The abi3-5mutant is one of the strongest abi3alleles,which all show reduced seed dormancy and decreased sensitivity to ABA during germination (Bies-Etheve et al.,1999).Seeds of the abi3-4and abi3-6mutants are nondormant,highly insensitive to ABA,and show reduced longevity and a high chlorophyll content similar to abi3-5.To study the suppression effect of the sua-1mutant on different abi3mutant alleles,double mutants were constructed.The ABA-insensitive abi3-4and abi3-6alleles,as well as the weak abi3-1and abi3-7alleles (Figure 5A),were combined with sua-1,sua-2,and sua-3.Surprisingly,none of these combinations showed any suppression phenotype,indicating that the suppres-sion of abi3-5by sua mutants is allele speciﬁc (Figure 5B).Detection of Functional ABI3Protein in the abi3-5sua-1Double MutantThe abi3-5mutation causes a frameshift leading to a premature stop codon after 34erroneous codons.The abi3-4mutant hasaFigure 3.SUA Is Expressed in All Tissues and Its Protein Is Localized in the Nucleus.(A)Quantitative real-time RT-PCR analysis of SUA expression in different tissues.SUA mRNA levels are normalized to ACTIN8mRNA levels.S6D,seedlings 6d after germination;R,roots;RL,rosette leaves;CL,cauline leaves;FB,ﬂower buds;S10to S20,siliques 10,12,14,16,18,and 20d after pollination.Data are from two independent biological replicates.Error bars represent SE .(B)Confocal analysis of subcellular localization of SUA:GFP in develop-ing embryo tissue from transgenic abi3-5sua plants containing the PSUA:SUA:GFP construct.Three nuclei with different GFP patterns are shown.Bar =2mM.Figure 4.SUA Interacts with U2AF 65.(A)Interaction between SUA and U2AF 65detected with the yeast two-hybrid assay.Cotransformed yeast strains were grown on SD-L-W-H with 5mM 3AT.Snf1and Snf4are yeast proteins that strongly interact (Jiang and Carlson,1997).(B)to (E)Interaction between SUA and U2AF 65based on FRET mea-sured by FLIM.FLIM analysis of protoplasts transiently expressing U2AF 65-CFP ([B]and [C])and coexpressing U2AF 65-CFP and SUA-YFP ([D]and [E]).Intensity channel ([B]and [D])and false color code ([C]and [E]).The absence of interaction results in a long lifetime,visible as a dark-blue color.Interaction leads to a reduction in donor lifetime,visible as a shift toward orange.A representative protoplast nucleus is shown.(F)Average CFP ﬂuorescence lifetime values for the FRET/FLIM analysis.N,number of nuclei analyzed.SUA Controls Alternative Splicing of ABI31939single nucleotide mutation that causes a stop codon at approx-imately the same position (Bies-Etheve et al.,1999;Figure 5A).Therefore,abi3-4and abi3-5produce ABI3transcripts that translate into truncated ABI3proteins with similar sizes.Never-theless,the phenotype of the abi3-5mutant is strongly sup-pressed by sua ,whereas that of abi3-4is not.To understand this discrepancy,we analyzed the ABI3protein in dry seeds of L er ,sua-1,abi3-4,abi3-5,and the double mutants abi3-4sua-1and abi3-5sua-1by immunoblotting.A speciﬁc antibody,targeted to the amino end of ABI3,was used for detection.The ABI3protein (720amino acids)migrates as a 116-kD polypeptide (Parcy et al.,1997).We detected two bands of approximately this size for the ABI3protein in L er and sua-1seeds.One of these two bands probably represents a modiﬁed version of ABI3.A truncated ABI3protein corresponding to a 428–amino acid polypeptide and migrating as a 70-kD band,was observed in the abi3-5mutant (Figure 6).A similar sized (416amino acids)highly abundant ABI3protein was found in abi3-4and abi3-4sua-1.The high abundance of the truncated ABI3protein in abi3-4seeds was previously observed by Parcy et al.(1997).In the abi3-5sua-1double mutant,two weak bands of comparable size to full-length ABI3were detected,along with the smaller truncated abi3-5mutant protein (Figure 6).The presence of full-length ABI3protein in abi3-5sua-1seeds was consistent with all the observed suppression phenotypes and predicts the pres-ence of an ABI3transcript with a restored reading frame that has lost the abi3-5premature stop codon.Identiﬁcation of a Novel ABI3Splice VariantThe abi3-5transcripts were analyzed in detail by RT-PCR and sequencing.In the abi3-5sua-1double mutant,we identiﬁed,besides the expected full-length abi3-5transcript,an alterna-tively spliced novel abi3-5transcript that lacks a cryptic intron of 77nucleotides.This cryptic intron is located shortly downstream of the abi3-5mutation and includes the premature abi3-5stop codon (Figure 5C).The combination of the 1-bp abi3-5deletion and the removal of the 77-nucleotide cryptic intron results in a transcript that restores the reading frame of abi3-5after 21erroneous and 26deleted codons.We named this transcript abi3-5-b and named the transcript with the retained intron abi3-5-a .The translated abi3-5-b polypeptide (abi3-5-b )is predicted to be 694amino acids.This protein contains all four ABI3protein domains (Figure 6B),and the phenotype of the abi3-5sua-1seeds indicated that abi3-5-b largely retains the ABI3molecular functions (Figure 1).The ABI3-b transcript only encodes a functional protein in the abi3-5mutant background.In the wild type,it causes a frame-shift and codes for a truncated protein of 429amino acids.This predicted truncated polypeptide was immunodetected in the sua-1single mutant and also,at lower levels,in wild-type L er seed protein extracts.The wild-type ABI3-b protein migrates with a similar speed in the gel as the proteins encoded by abi3-4(a and b splicing forms)and abi3-5(a splicing form)mutants (Figure 6).In addition to the accumulation of the ABI3-b splice variant,the sua-1mutant also shows an overall increase in ABI3expression.The amount of ABI3-a transcript,coding for full-length ABI3,is higher in sua-1than in the wild type (Figure 7A).This could explain the increased ABA sensitivity of sua-1seeds.Instead,overall ABI3expression in sua-2seeds is similar to that in wild-type Col-0,but the portion of the transcript coding for full-length ABI3is reduced,resulting in a decrease of ABA sensitivity (Figure 1E).We tested the possibility that sua-1has again-of-functionFigure 5.sua Is an Allele-Speciﬁc Suppressor of the abi3-5Allele.(A)Schematic structure of the ABI3gene.The locations and nature of the abi3-1,abi3-4,abi3-5,abi3-6,and abi3-7mutations are indicated.UTRs are shown in white,exons in gray,and introns as thick lines.The box with diagonal stripes represents the cryptic intron.(B)Table showing the suppression of the abi3phenotype in different combinations of sua and abi3mutant alleles.A check mark indicates abi3suppression;the “x”indicates absence of abi3suppression.ND,not determined.(C)Sequence of the ABI3cryptic intron and surrounding region.The asterisk indicates the single base pair deleted in abi3-5,and the subsequent stop codon is underlined.The cryptic intron is shown in lowercase letters.1940The Plant Celleffect on ABI3expression by transforming the sua-1mutant allele,expressed from the endogenous SUA promoter,into the sua-2mutant.Indeed,the obtained transformants showed a higher ABI3expression than the sua-2mutant and had increased ABA sensitivity (see Supplemental Figure 1online).This indicates that sua-1is a gain-of-function mutant regarding ABI3expres-sion.ABI3Alternative Splicing Is Developmentally Regulated The relative abundance of ABI3-a and ABI3-b transcripts was quantiﬁed in wild-type seeds by real-time RT-PCR.Developing siliques 16d after pollination showed a very low abundance of ABI3-b transcript in L er and Col-0(1.5361.36%and 0.9560.83%,respectively,of the overall ABI3transcripts;Figure 7A).During progressive development of wild-type siliques,the ratiobetween both ABI3transcripts shifted toward ABI3-b .At 20d after pollination,the amount of ABI3-b exceeded that of ABI3-a (Figure 7B).The observed change in ratio between ABI3-a and ABI3-b transcripts during seed maturation indicates that alter-native splicing of ABI3is developmentally regulated.DISCUSSIONABI3Is Regulated by Alternative SplicingThe transcription factor ABI3regulates seed maturation and inﬂuences seed quality.The abundance of ABI3is tightly regu-lated at different levels.In addition to complex genetic interac-tions with LEC1,LEC2,and FUS3at the transcriptional level (To et al.,2006),ABI3expression is controlled posttranscriptionally.Alternative splicing of ABI3homologs in cereal species (Triticum aestivum and Oryza sativa )and dicots (Pisum sativum )(McKibbinFigure 6.Detection of Full-Length ABI3Protein in the abi3-5sua-1Double Mutant.(A)Immunoblot analysis of ABI3protein.Total protein was extracted from freshly harvested seeds and separated on a Tris-Gly SDS 4to 12%polyacrylamide gradient gel.The ABI3protein is identiﬁed as a double band of ;116kD in L er ,sua-1,and abi3-5sua-1.The truncated ABI3proteins (D ABI3)produced by abi3-4,abi3-5,and abi3-5sua-1and the novel splicing variant of ABI3are ;70kD.Asterisk indicates a nonspe-ciﬁc band that is used as loading control.Sizes of the molecular markers (in kilodaltons)are shown next to the blot.(B)Predicted ABI3protein isoforms.Gray boxes represent the con-served functional motifs of ABI3(from left to right:A1,B1,B2,and B3).Boxes with diagonal stripes represent erroneous amino acidsstretches.Figure 7.Quantiﬁcation of ABI3Splicing Variants.Quantitative real-time RT-PCR analysis of ABI3-a (white)and ABI3-b (gray)expression in L er ,sua-1,abi3-5,abi3-5sua-1,Col-0,and sua-2(A)and in L er developing siliques 10to 20d after pollination (DAP)(B).For (A),mRNA was extracted from siliques 16d after pollination.ABI3mRNA levels are normalized to ACTIN8mRNA levels.Data are from two independent biological replicates.Error bars represent SE .SUA Controls Alternative Splicing of ABI31941et al.,2002;Fan et al.,2007;Gagete et al.,2009)generates multiple mis-spliced transcripts that often code for truncated polypeptides.This has been linked to reduced grain quality in rice and wheat(McKibbin et al.,2002;Fan et al.,2007).Here,we show that the ABI3gene of Arabidopsis is also regulated by alternative splicing.A77-bp cryptic ABI3intron is alternatively spliced,which leads to the occurrence of two transcripts.The ABI3-a transcript encodes a full-length ABI3protein,and the ABI3-b transcript encodes a truncated protein that contains two of the four functional domains.Splicing of the cryptic intron of ABI3is developmentally regulated,and ABI3-b accumulates only at the end of seed maturation.This probably contributes to a fast downregulation of full-length ABI3in ripe seeds,which is neces-sary to inhibit the seed maturation program in germinating seeds. Transcripts with a long39untranslated region(UTR)or with 39UTR-located introns can be detected and degraded by the nonsense-mediated decay machinery in plants(Kere´nyi et al., 2008).To distinguish a natural stop codon from a premature stop codon,nonsense-mediated decay requires a second signal that has not been identiﬁed yet in plants(van Hoof and Green,2006). The ABI3-b transcript contains a premature stop codon but probably lacks this second signal because it is not affected by nonsense-mediated decay.The protein encoded by the ABI3-b transcript contains the A1acidic transcriptional activation do-main and theﬁrst basic domain and might still mediate ABA signaling during late seed maturation.The prevalent model of splicing in Arabidopsis is intron deﬁ-nition,in which intronic sequences are recognized by the spliceosomal complex.The features of a canonical plant intron are a consensus59splice site(AG/GU,where GU is the more conserved dinucleotide),a U-rich sequence,and a consensus 39splice site(CAG/G where AG is invariant)(Simpson and Filipowicz,1996;Lorkovic´et al.,2000).Arabidopsis exons con-tain on average29%U,while introns have on average42%U (Reddy,2007).It was shown that U-rich elements can function as splicing signals(Simpson et al.,2004),and short introns and introns with low AU content are more likely to be retained(Wang and Brendel,2006).The ABI3cryptic intron has sequence similarities with canonical plant introns,in particular with the consensus sequences at the two borders(Figure5C),but it has a U content of only29%,while the other ABI3introns have,on average,46%U.Because of that,the cryptic ABI3intron may not be easily recognized by the spliceosomal complex.SUA Controls Alternative Splicing of ABI3SUA suppresses splicing of the cryptic ABI3intron and thereby inﬂuences the ratio between the ABI3-a and ABI3-b transcripts. Reduced suppression of the cryptic intron in the sua mutant leads to an increased amount of ABI3-b transcript and de-creased levels of the ABI3-a transcript.However,a substantial amount of ABI3-a transcript could still be detected in the sua mutant.Other splicing factors probably act redundantly with SUA in the suppression of the cryptic ABI3intron.Alternative splicing in plants is regulated by tissue-speciﬁc developmental cues and stresses and might provide a means for optimal adaptation to the environment(Ali and Reddy,2008). Alternative splicing of ABI3could also be regulated by speciﬁc environmental conditions.In this respect,it is interesting to note that publicly available microarray data show an upregulation of SUA expression by senescence(Zimmermann et al.,2004).The water content of seeds strongly decreases during the maturation phase until;7%in mature seed(Baud et al.,2002).This process is comparable to senescence and also coincides with increased SUA mRNA levels(Figure3A).Higher SUA abundance will favor cryptic intron retention and increase the full-length ABI3protein levels during seed maturation.Consistent with that,our exper-iments showed a correlation between increased levels of SUA transcript and a reduction in ABI3-b levels in transgenic plants. The abi3-5sua-1PSUA:SUA:GFP#8line,for instance,showed increased levels of SUA transcript and reduced amounts of abi3-5-b,resulting in an enhanced abi3-5phenotype(see Supple-mental Figure2online;Figures1A and1D).SUA-mediated alternative splicing of ABI3could represent a system toﬁne-tune seed maturation.However,in wild-type plants,the ABI3-b transcript accumulates at the end of seed maturation when SUA is still substantially expressed.Possibly,the SUA protein is not active or degraded at the end of seed maturation.Alternatively, other factors could counteract the role of SUA in retention of the cryptic intron at this time.The sua-1single mutant showed increased ABA sensitivity during germination.This is probably caused by upregulation of ABI3expression,which does not occur in sua-2.This difference between sua-1(in a L er background)and sua-2(in a Col-0 background)could be explained by natural genetic variation between L er and Col-0that modiﬁes the sua mutant phenotype. However,sua-2plants transformed with sua-1also showed an increased ABI3expression and enhanced ABA sensitivity. Therefore,it is more likely that sua-1is a gain-of-function allele, which is translated into a truncated protein.This predicted polypeptide includes the RNA recognition motifs and the Zn ﬁnger motif but lacks the G patch domain at the C terminus.The nonfunctional mutant sua-1protein might compete for sub-strates with other proteins of the mRNA splicing machinery and could therefore function as a dominant-negative allele.The abi3-5mutant still contains a small amount of abi3-5-b transcript,which encodes a functional ABI3protein.Conse-quently,abi3-5is not a complete loss-of-function mutant.The sua mutant can suppress abi3-5because it enhances the amount of abi3-5-b transcript.SUA Has a Conserved Role in SplicingThe conserved domain architecture of SUA and its role in the suppression of the cryptic ABI3intron indicate a function in mRNA processing.Moreover,the speckledﬂuorescence pat-terns observed in nuclei expressing the chimeric SUA:GFP gene are similar to those obtained with Ser/Arg-rich GFP proteins, which are involved in RNA metabolism in plants(Lorkovic´and Barta,2004).The SUA protein has two RNA recognition motifs, which are also found in many eukaryotic RNA processing pro-teins(Burd and Dreyfuss,1994).Based on its functional motifs, SUA could bind directly to speciﬁc RNA targets.However,SUA might also interact with the mRNA targets indirectly and be part of the spliceosome,which is composed of;300proteins in Arabidopsis(Reddy,2007).1942The Plant Cell。

NATURE RNA-Seq a revolutionary tool for transcriptomics

The transcriptome is the complete set of transcripts in a cell, and their quantity, for a specific developmental stage or physi-ological condition. Understanding the transcriptome is essential for interpreting the functional elements of the genome and revealing the molecular constituents of cells and tissues, and also for understand-ing development and disease. The key aims of transcriptomics are: to catalogue all species of transcript, including mRNAs, non-coding RNAs and small RNAs; to determine the transcriptional structureof genes, in terms of their start sites, 5′and 3′ ends, splicing patterns and other post-transcriptional modifications; and to quantify the changing expression levels of each transcript during development and under different conditions.Various technologies have been developed to deduce and quantify the transcriptome, including hybridization- or sequence-based approaches. Hybridization-based approaches typically involve incubating fluorescently labelled cDNA with custom-made microarrays or commercial high-density oligo microar-rays. Specialized microarrays have also been designed; for example, arrays with probes spanning exon junctions canbe used to detect and quantify distinct spliced isoforms1. Genomic tiling microar-rays that represent the genome at high density have been constructed and allow the mapping of transcribed regions to avery high resolution, from several basepairs to ~100 bp2–5. Hybridization-basedapproaches are high throughput andrelatively inexpensive, except for high-resolution tiling arrays that interrogatelarge genomes. However, these methodshave several limitations, which include:reliance upon existing knowledge aboutgenome sequence; high background levelsowing to cross-hybridization6,7; and alimited dynamic range of detection owingto both background and saturation ofsignals. Moreover, comparing expressionlevels across different experiments is oftendifficult and can require complicatednormalization methods.In contrast to microarray methods,sequence-based approaches directly deter-mine the cDNA sequence. Initially, Sangersequencing of cDNA or EST libraries8,9was used, but this approach is relativelylow throughput, expensive and generallynot quantitative. Tag-based methods weredeveloped to overcome these limitations,including serial analysis of gene expression(SAGE)10,11, cap analysis of gene expression(CAGE)12–14 and massively parallel signaturesequencing (MPSS)15–17. These tag-basedsequencing approaches are high through-put and can provide precise, ‘digital’ geneexpression levels. However, most arebased on expensive Sanger sequencingtechnology, and a significant portion ofthe short tags cannot be uniquely mappedto the reference genome. Moreover, onlya portion of the transcript is analysed andisoforms are generally indistinguishablefrom each other. These disadvantageslimit the use of traditional sequencingtechnology in annotating the structure oftranscriptomes.Recently, the development of novelhigh-throughput DNA sequencing meth-ods has provided a new method for bothmapping and quantifying transcriptomes.This method, termed RNA-Seq (RNAsequencing), has clear advantages overexisting approaches and is expected to rev-olutionize the manner in which eukaryotictranscriptomes are analysed. It has alreadybeen applied to Saccharomyces cerevisiae,Schizosaccharomyces pombe, Arabidopsisthaliana, mouse and human cells18–24. Here,we explain how RNA-Seq works, discussits challenges and provide an overview ofstudies that have used this approach, whichhave already begun to change our view ofeukaryotic transcriptomes.RNA-Seq technology and benefitsRNA-Seq uses recently developed deep-sequencing technologies. In general, apopulation of RNA (total or fractionated,such as poly(A)+) is converted to a libraryof cDNA fragments with adaptors attachedto one or both ends (FIG. 1). Each molecule,with or without amplification, is thensequenced in a high-throughput mannerto obtain short sequences from one end(single-end sequencing) or both ends(pair-end sequencing).The reads are typi-cally 30–400 bp, depending on the DNA-sequencing technology used. In principle,any high-throughput sequencing technol-ogy25 can be used for RNA-Seq, and theIllumina IG18–21,23,24, Applied BiosystemsSOLiD22 and Roche 454 Life Science26–28I N N OVAT I O NRNA-Seq: a revolutionary tool fortranscriptomicsZhong Wang, Mark Gerstein and Michael SnyderAbstract | RNA-Seq is a recently developed approach to transcriptome profilingthat uses deep-sequencing technologies. Studies using this method havealready altered our view of the extent and complexity of eukaryotictranscriptomes. RNA-Seq also provides a far more precise measurement oflevels of transcripts and their isoforms than other methods. This article describesthe RNA-Seq approach, the challenges associated with its application, and theadvances made so far in characterizing several eukaryote transcriptomes.RNA-Seq […] is expectedto revolutionize themanner in which eukaryotictranscriptomes are analysed.NATURE REVIEwS |genetics VOLUME 10 | jANUARy 2009 |57PeRSPecTiveS© 2009 Macmillan Publishers Limited. All rights reservedsystems have already been applied for this purpose. The Helicos Biosciences tSMS system has not yet been used for published RNA-Seq studies, but is also appropriate and has the added advantage of avoiding amplification of target cDNA. Following sequencing, the resulting reads are either aligned to a reference genome or reference transcripts, or assembledde novo without the genomic sequenceto produce a genome-scale transcription map that consists of both the transcrip-tional structure and/or level of expression for each gene.Although RNA-Seq is still a technology under active development, it offers several key advantages over existing technologies (Table 1).First, unlike hybridization-based approaches, RNA-Seq is not limited to detecting transcripts that correspondto existing genomic sequence. For example, 454-based RNA-Seq has beenused to sequence the transcriptome ofthe Glanville fritillary butterfly27. Thismakes RNA-Seq particularly attractivefor non-model organisms with genomicsequences that are yet to be determined.RNA-Seq can reveal the precise locationof transcription boundaries, to a single-base resolution. Furthermore, 30-bp shortreads from RNA-Seq give informationabout how two exons are connected,whereas longer reads or pair-end shortreads should reveal connectivity betweenmultiple exons. These factors make RNA-Seq useful for studying complex tran-scriptomes. In addition, RNA-Seq can alsoreveal sequence variations (for example,SNPs) in the transcribed regions22,24.A second advantage of RNA-Seqrelative to DNA microarrays is thatRNA-Seq has very low, if any, backgroundsignal because DNA sequences canbeen unambiguously mapped to uniqueregions of the genome. RNA-Seq doesnot have an upper limit for quantifica-tion, which correlates with the numberof sequences obtained. Consequently,it has a large dynamic range of expres-sion levels over which transcripts can bedetected: a greater than 9,000-fold rangewas estimated in a study that analysed 16million mapped reads in Saccharomycescerevisiae18, and a range spanning fiveorders of magnitude was estimated for40 million mouse sequence reads20. Bycontrast, DNA microarrays lack sensitivityfor genes expressed either at low or veryhigh levels and therefore have a muchsmaller dynamic range (one-hundredfoldto a few-hundredfold) (FIG. 2). RNA-Seqhas also been shown to be highly accuratefor quantifying expression levels, as deter-mined using quantitative PCR (qPCR)18 andspike-in RNa controls of known concentra-tion20. The results of RNA-Seq also showhigh levels of reproducibility, for bothtechnical and biological replicates18,22.Finally, because there are no cloning steps,and with the Helicos technology there isno amplification step, RNA-Seq requiresless RNA sample.Taking all of these advantages intoaccount, RNA-Seq is the first sequencing-based method that allows the entiretranscriptome to be surveyed in a veryhigh-throughput and quantitative man-ner. This method offers both single-baseresolution for annotation and ‘digital’gene expression levels at the genome scale,often at a much lower cost than eithertiling arrays or large-scale Sanger ESTsequencing.Challenges for RNA-SeqLibrary construction.The ideal methodfor transcriptomics should be able todirectly identify and quantify all RNAs,small or large. Although there are onlya few steps in RNA-Seq (FIG. 1), it doesinvolve several manipulation stages dur-ing the production of cDNA libraries,which can complicate its use in profilingall types of transcript.Unlike small RNAs (microRNas(miRNAs), Piwi-interacting RNas (piRNAs),short interfering RNas (siRNAs)and manyothers), which can be directly sequencedafter adaptor ligation, larger RNA mol-ecules must be fragmented into smallerpieces (200–500 bp) to be compatiblewith most deep-sequencing technologies.Common fragmentation methods includeCoding sequenceORFP e r s P e c t i v e s58 | jANUARy 2009 | VOLUME 10 /reviews/genetics© 2009 Macmillan Publishers Limited. All rights reservedRNA fragmentation (RNA hydrolysis or nebulization) and cDNA fragmentation (DNase I treatment or sonication). Each of these methods creates a different bias in the outcome. For example, RNA fragmen-tation has little bias over the transcript body20, but is depleted for transcript ends compared with other methods (FIG. 3). Conversely, cDNA fragmentation is usually strongly biased towards the iden-tification of sequences from the 3′ ends of transcripts, and thereby provides valuable information about the precise identity of these ends18 (FIG. 4).Some manipulations during library construction also complicate the analysis of RNA-Seq results. For example, many shorts reads that are identical to each other can be obtained from cDNA librariesthat have been amplified. These could bea genuine reflection of abundant RNAspecies, or they could be PCR artefacts.One way to discriminate between thesepossibilities is to determine whether thesame sequences are observed in differentbiological replicates.Another key consideration concerninglibrary construction is whether or not toprepare strand-specific libraries, as hasbeen done in two studies21,22. These librarieshave the advantage of yielding informationabout the orientation of transcripts, whichis valuable for transcriptome annotation,especially for regions with overlappingtranscription from opposite directions2,19,29;however, strand-specific libraries arecurrently laborious to produce because theyrequire many steps22 or direct RNA–RNAligation21, which is inefficient. Moreover,it is essential to ensure that the antisensetranscripts are not artefacts of reverse tran-scription30. Because of these complications,most studies thus far have analysed cDNAswithout strand information.Bioinformatic challenges.Like otherhigh-throughput sequencing technolo-gies, RNA-Seq faces several informaticschallenges, including the development ofefficient methods to store, retrieve andprocess large amounts of data, which mustbe overcome to reduce errors in imageanalysis and base-calling and removelow-quality reads.Table 1 |Advantages of RNA-Seq compared with other transcriptomics methodsP e r s P e c t i v e sOnce high-quality reads have been obtained, the first task of data analysis is to map the short reads from RNA-Seq to the reference genome, or to assemble them into contigs before aligning them to the genomic sequence to reveal transcription structure. There are several programs for mapping reads to the genome, including ELAND, SOAP31, MAQ32 and RMAP33 (information about these can be found atthe Illumina forum and at SEQanswers).However, short transcriptomic reads also only needs to be given to poly(A) tailsand to a small number of exon–exonjunctions. Poly(A) tails can be identifiedsimply by the presence of multiple As orTs at the end of some reads. Exon–exonjunctions can be identified by the pres-ence of a specific sequence context (theGT–AG dinucleotides that flank splicesites) and confirmed by the low expressionof intronic sequences, which are removedduring splicing. Transcriptome mapsa junction library that contains all theknown and predicted junction sequencesand map reads to this library19,20. A chal-lenge for the future is to develop computa-tionally simple methods to identify novelsplicing events that take place between twodistant sequences or between exons fromtwo different genes.For large transcriptomes, alignmentis also complicated by the fact that a sig-nificant portion of sequence reads matchP e r s P e c t i v e sgenes are presumably either not expressed under this condition (for example, sporu-lation genes18) or do not have poly(A) tails. Analyzing many different conditions can further increase the coverage; inS. pombe 122 million reads from six differ-ent growth conditions detected transcrip-tion from >99% of annotated genes19.In general, the larger the genome, the more complex the transcriptome, the more sequencing depth is required for adequate coverage. Unlike genome-sequencing cov-erage, it is less straightforward to calculate the coverage of the transcriptome; thisis because the true number and level of different transcript isoforms is not usually known and because transcription activity varies greatly across the genome. One study used the number of unique transcription start sites as a measure of coverage in mouse embryonic cells, and demonstrated that at 80 million reads, the number of start sites reached a plateau22 (FIG. 5b). However, this approach does not address transcrip-tome complexity in alternative splicing and transcription termination sites; presumably further sequencing can reveal additionalvariants.New transcriptomic insights Despite the challenges described above, the advantages of RNA-Seq have enabled us to generate an unprecedented global view of the transcriptome and its organi-zation for a number of species and cell types. Before the advent of RNA-Seq,it was known that a much greater than expected fraction of the yeast, Drosophila melanogaster and human genomes are transcribed2,4,36, and for yeast and humans a number of distinct isoforms have been found for many genes2,4. However, the starts and ends of most transcripts and exons had not been precisely resolved and the extent of spliced heterogeneity remained poorly understood. RNA-Seq, with its high resolution and sensitivity has revealed many novel transcribed regions and splicing isoforms of known genes, and has mapped 5′ and 3′ boundaries for many genes.Mapping gene and exon boundaries. The single-base resolution of RNA-Seq has the potential to revise many aspectsof the existing gene annotation, including gene boundaries and introns for known genes as well as the identification of novel transcribed regions. 5′ and 3′ boundaries can be mapped to within 10–50 bases by a precipitous drop in signal. 3′ boundaries can be precisely mapped by searching forpoly(A) tags, and introns can be mappedby searching for tags that span GT–AGsplicing consensus sites. Using these meth-ods the 5′ and 3′ boundaries of 80% and85% of all annotated genes, respectively,were mapped in S. cerevisiae18. Similarly,in S. pombe many boundaries were definedby RNA-Seq data in combination withtiling array data19.These two studies led to the discoveryof many 5′ and 3′ UTRs that had notbeen analysed previously. In S. cerevisiae,extensive 3′-end heterogeneity wasdiscovered at two levels: first, localheterogeneity exists in which a cluster ofsites are involved, typically within a 10 bpwindow; second, there are distinct regionsof poly(A) addition for 540 genes (FIG. 4).It is plausible that these different 3′ endsconfer distinct properties to the differentmRNA isoforms, such as mRNA localiza-tion or degradation signals, which in turnmight be responsible for unique biologicalfunctions18,19. In addition to 3′ heterogene-ity, the list of upstream ORFs within the 5′UTRs of mRNAs (uORFs) was also greatlyexpanded from 17 to 340 (6% of yeastgenes)18; uORFs regulate mRNA transla-tion37 or stability38, so these sequencesmight make a previously underappreciatedcontribution to the regulatory sophistica-tion of eukaryotic genomes. Interestingly,many mRNAs with uORFs are transcrip-tion factors, suggesting that these regulatorsare themselves heavily regulated.The mapping of transcript boundariesrevealed several novel features of eukaryo-tic gene organization. Many yeast geneswere found to overlap at their 3′ ends18.Using relaxed criteria similar to thoseemployed in a recent study18 we found that808 pairs, approximately 25% of all yeastORFs, overlap at their 3′ ends18. Likewise,antisense expression is enriched in the 3′exons of mouse transcripts22. These featuresmight confer interesting regulatory proper-ties on the affected genes. For multicellularorganisms, antisense transcription couldmodulate gene expression through theproduction of siRNAs or through dsRNaediting39,40. For yeast, which seems to lacksiRNA and dsRNA-editing functions,transcription from one gene might interferewith that from an overlapping gene, orcoordinate gene expression through othermechanisms.Extensive transcript complexity.RNA-Seqcan be used to quantitatively examinesplicing diversity by searching for readsthat span known splice junctions as wellas potential new ones. In humans, 31,618known splicing events were confirmed(11% of all known splicing events) and 379novel splicing events were discovered24.ture Reviews |Genetics′Local heterogeneityDistinct poly(A) sitesFigure 4 | Poly(A) tags from RnA-seq. A region containing two overlapping transcripts (ACT1, from the actin gene, and YFL040W, an uncharacterized ORF) from the Saccharomyces cerevisiae genome is shown. Arrows point to transcription direction. The poly(A) tags from RNA-Seq experiments are shown below these transcripts, with arrows indicating transcription direction. The precise location of each locus identified by poly(A) tags reveals the heterogeneity in poly(A) sites, for example, ACT1 has two big clusters, both with a few bases of local heterogeneity. The transcription direction revealed by poly(A) tags also helps to resolve 3′-end overlapping transcribed regions18.P e r s P e c t i v e sNATURE REVIEwS |genetics VOLUME 10 | jANUARy 2009 |61© 2009 Macmillan Publishers Limited. All rights reservedglossaryCap analysis of gene expression(CaGe). Similar to SaGe, except that 5′-end information of the transcript is analysed instead of 3′-end information. ContigsA group of sequences representing overlapping regions from a genome or transcriptome.dsRNA editingSite-specific modification of a pre-mRNa by dsRNa-specific enzymes that leads to the production of variant mRNa from the same gene.Genomic tiling microarraya DNa microarray that uses a set of overlapping oligonucleotide probes that represent a subset of or the whole genome at very high resolution.Massively parallel signature sequencing (MPSS). a gene expression quantification method that determines 17–20-bp ‘signatures’ from the ends of a cDNa molecule using multiple cycles of enzymatic cleavage and ligation.MicroRNA(miRNa). Small RNa molecules that areprocessed from small hairpin RNa (shRNa)precursors that are produced from miRNagenes. miRNas are 21–23 nucleotides in lengthand through the RNa-induced silencing complexthey target and silence mRNas containing imperfectlycomplementary sequence.Piwi-interacting RNAs(piRNa). Small RNa species that are processedfrom single-stranded precursor RNas. Theyare 25–35 nucleotides in length and formcomplexes with the piwi protein. piRNas areprobably involved in transposon silencing andstem-cell function.Quantitative PCR(qPCR). an application of PCR to determinethe quantity of DNa or RNa in a sample. Themeasurements are often made in real time andthe method is also called real-time PCR.Sequencing depthThe total number of all the sequences reads or basepairs represented in a single sequencing experiment orseries of experiments.Serial analysis of gene expression(SaGe). a method that uses short ~14–20-bp sequencetags from the 3′ ends of transcripts to measure geneexpression levels.Short interfering RNA(siRNa). RNa molecules that are 21–23 nucleotides longand that are processed from long double-stranded RNas;they are functional components of the RNai-inducedsilencing complex. siRNas typically target and silencemRNas by binding perfectly complementary sequencesin the mRNa and causing their degradation and/ortranslation inhibition.Spike-in RNAa few species of RNa with known sequence and quantitythat are added as internal controls in RNa-Seq experiments.62 | jANUARy 2009 | VOLUME 10 /reviews/genetics© 2009 Macmillan Publishers Limited. All rights reservedDefining transcription levelAs RNA-Seq is quantitative, it can be used to determine RNA expression levels more accurately than microarrays. In principle, it is possible to determine the absolute quantity of every molecule in a cell population, and directly compare results between experiments. Several methods have been used for quantification. For RNA fragmentation followed by cDNA synthesis, which gives more uniform cov-erage of each exon, gene expression levels can be deduced from the total numberof reads that fall into the exons of a gene, normalized by the length of exons that can be uniquely mapped24; for 3′-biased methods, read counts from a window near the 3′ end are used18. Gene expression levels determined by these methods closely correlate with qPCR and RNA spike-in controls.One particularly powerful advantage of RNA-Seq is that it can capture transcrip-tome dynamics across different tissues or conditions without sophisticated normali-zation of data sets19,20,22. RNA-Seq has been used to accurately monitor gene expres-sion during yeast vegetative growth18, yeast meiosis19 and mouse embryonic stem-cell differentiation22, to track gene expression changes during development, and to provide a ‘digital measurement’ of gene expression difference between differ-ent tissues20. Because of these advantages, RNA-Seq will undoubtedly be valuable for understanding transcriptomic dynamics during development and normal physi-ological changes, and in the analysis of biomedical samples, where it will allow robust comparison between diseased and normal tissues, as well as the subclassification of disease states.Future directionsAlthough RNA-Seq is still in the early stages of use, it has clear advantages over previously developed transcriptomic methods. The next big challenge for RNA-Seq is to target more complex transcriptomes to identify and track the expression changes of rare RNA isoforms from all genes. Technologies that will advance achievement of this goal are pair-end sequencing, strand-specific sequencing and the use of longer reads to increase coverage and depth. As the cost of sequencing continues to fall, RNA-Seq is expected to replace microarrays for many applications that involve determin-ing the structure and dynamics of the transcriptome.Zhong Wang and Michael Snyder are at the Departmentof Molecular, Cellular and Developmental Biology, andMark Gerstein is at the Department of Molecular,Biophysics and Biochemistry, Yale University, 219Prospect Street, New Haven, Connecticut 06520, USA.Correspondence to M.S.e‑mail: michael.snyder@doi:10.1038/nrg2484Published online 18 November 20081. Clark, T. A., Sugnet, C. W. & Ares, M. Jr.Genomewide analysis of mRNA processing in yeastusing splicing-specific microarrays. Science296,907–910 (2002).2. David, L. et al. A high-resolution map of transcriptionin the yeast genome. Proc. Natl Acad. Sci. USA103,5320–5325 (2006).3. Yamada, K. et al. Empirical analysis of transcriptionalactivity in the Arabidopsis genome. Science 302,842–846 (2003).4. Bertone, P. et al. 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Q., et al. Systematic analysis of transcribed lociin ENCODE regions using RACE sequencing revealsextensive transcription in the human genome. GenomeBiol.9, R3 (2008).31. Li, R., Li, Y., Kristiansen, K. & Wang, J. SOAP: shortoligonucleotide alignment program. Bioinformatics24, 713–714 (2008).32. Li, H., Ruan, J. & Durbin, R. Mapping short DNAsequencing reads and calling variants using mappingquality scores. Genome Res. 19 Aug 2008(doi:10.1101/gr.078212.108).33. Smith, A. D., Xuan, Z. & Zhang, M. Q. Usingquality scores and longer reads improves accuracyof Solexa read mapping. BMC Bioinformatics9,128 (2008).34. Hillier, L. W. et al. Whole-genome sequencing andvariant discovery in C. elegans. Nature Methods5,183–188 (2008).35. Campbell, P. J. et al. Identification of somaticallyacquired rearrangements in cancer using genome-widemassively parallel paired-end sequencing. NatureGenet.40, 722–729 (2008).36. Manak, J. R. et al. 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基于TaqMan_探针的实时荧光LAMP_检测肉制品中鸭源性成分研究

第46卷第6期2023年11月河北农业大学学报JOURNAL OF HEBEI AGRICULTURAL UNIVERSITYVol.46 No.6Nov.2023基于TaqMan探针的实时荧光LAMP 检测肉制品中鸭源性成分研究柳梦思1，时国强1，高娜婷1，焦义然1，孙旭飞2，孟亚南3，董振国1（1．河北三狮生物科技有限公司，河北石家庄 050035；2．河北农业大学食品科技学院，河北保定 071000；3．河北农业大学生命科学学院，河北保定 071000）摘要：本研究建立1种基于TaqMan探针的实时荧光环介导等温扩增（Loop-mediated isothermal amplification,LAMP）（TaqMan—LAMP）检测方法，用于肉制品中鸭源性成分的快速鉴别。

以鸭线粒体cytb基因的保守序列设计特异性引物，优化并建立基于TaqMan探针的实时荧光LAMP检测方法，对该方法进行特异性、灵敏度试验；同时与《GB/T38164—2019常见畜禽动物源成分检测方法实时荧光PCR法》作比较，对市售肉制品进行检测，验证本方法的准确性。

所建立的TaqMan-LAMP检测方法特异性强，仅鸭肉基因组DNA呈现特异性扩增曲线，30 min内完成检测，灵敏度为0.001%（w/w），重复性好，批次内变异系数CV为1.99%，对47份肉制品进行检测，检测结果与国标法相比，相对敏感性、特异性和符合率均为100%。

本研究建立的鸭源性成分TaqMan-LAMP检测方法检测快速、特异性强、灵敏度高、重复性好，适用于肉制品中鸭源性成分的快速检测。

关键词：环介导等温扩增；TaqMan-LAMP；鸭源性成分；检测中图分类号：S152开放科学（资源服务）标识码（OSID）：文献标志码：AStudy on detection of duck-derived ingredients in meat products by real-time fluorescent LAMP based on TaqMan probeLIU Mengsi1, SHI Guoqiang1, GAO Nating1, JIAO Yiran1, SUN Xufei2, MENG Yanan3, DONG Zhenguo1(1. Hebei sanshibio-tech co.,ltd, Shijiazhuang 050035, China;2. College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China;3. College of Life Sciences, Hebei Agricultural University, Baoding 071000, China)Abstract: In this study, a real-time Loop-mediated isothermal amplification assay based on TaqMan probe(TaqMan-LAMP) was developed for the rapid identification of duck-derived components in meat products.Cytbwas used as the target gene to design specific primers followed by establishment and optimization of a real-timefluorescent LAMP detection method whose specificity and sensitivity was then tested and compared with GB/T38164-2019 real-time fluorescence PCR method on the commercial meat products. Only the genomic DNA ofduck meat showed a specific amplification curve within 30 min with a sensitivity of 0.001% (w/w), suggesting thehigh specificity of the TagMan-LAMP repeatability of the method was high, and the coefficient of variation (CV )收稿日期：2023-09-23基金项目：国家自然科学基金项目（32172288）；石家庄市科技孵化计划项目（211510109A）.第一作者：柳梦思（1989—），硕士，从事合成化学在分子生物学中的应用等研究.E-mail：********************通信作者：孟亚南（1992—），博士，副教授，从事微生物核酸快速检测等研究.E-mail：*******************文章编号：1000-1573(2023)06-0089-06DOI：10.13320/ki.jauh.2023.009790第46卷河北农业大学学报in batches was 1.99%. The detection results of 47 meat products were compared with the national standard method, and the relative sensitivity, specificity and coincidence rate were 100%.The TaqMan-LAMP detection method established in this study is rapid with strong specificity, high sensitivity and good reproducibility. Thus, the Tag-LAMP method is is suitable for rapid detection of duck-derived components in meat products.Keywords: Loop-mediated isothermal amplification, TaqMan -LAMP, duck-derived ingredients, detection法GB/T 38164-2019［25］进行验证。

Terminator 5′-Phosphate-Dependent Exonuclease 产品说明

ManualTerminator 5′-Phosphate-Dependent ExonucleaseF or Research Use Only. Not for use in diagnostic procedures.Contents1. Introduction (3)2. Product designations and kit components (3)3. Product specifications (3)4. General considerations (3)5. Standard procedure (5)6. Alternative procedure (5)7. Further support (7)1. IntroductionTerminator 5′-Phosphate-Dependent Exonuclease is a processive 5′→3′ exonuclease that digests RNA having a 5′ monophosphate. It does not digest RNA having a 5′-triphosphate, 5′-cap or 5′-hydroxyl group. These enzymatic properties make Terminator Exonuclease ideal for producing mRNA-enriched samples from both eukaryotic and prokaryotic total RNA preparations by selectively digesting the ribosomal RNA (rRNA).Terminator Exonuclease can be used to isolate eukaryotic mRNA substantially free of 18S and 28S rRNA without using an oligo(dT) matrix. Further, Terminator Exonuclease provides a simple andeffective method for purifying unprocessed, primary bacterial transcripts by removing large rRNA and other processed transcripts.Terminator Exonuclease will also digest single-stranded DNA (ssDNA) having a 5′-phosphate group. It does not digest ssDNA or dsDNA having a 5′-triphosphate or a 5′-hydroxyl group. Terminator Exonuclease is not inhibited by RNase inhibitors such as RNasin (Promega Corporation), Prime RNase Inhibitor (Eppendorf AG) or RiboGuard™ RNase Inhibitor.Terminator 5′-Phosphate-Dependent Exonuclease is available in a 40 unit size at a concentration of 1 U/µL. Two different Terminator 10X Reaction Buffers are also provided with the enzyme, each optimised for different applications.2. Product designations and kit componentsProductKit size Reagent description Catalogue number Part number Volume 3. Product specificationsStorage: Store only at -20 °C in a freezer without a defrost cycle.Storage buffer: Terminator Exonuclease is supplied in a 50% glycerol solution containing 50 mM Tris-HCl (pH 7.5), 0.1 M NaCl, 0.1 mM EDTA, 1 mM dithiothreitol and 0.1% Triton X-100.Unit definition: One unit digests 1 μg of rRNA substrate into acid-soluble nucleotides in 60 minutes at 30 °C under standard assay conditions.Quality control: Terminator Exonuclease is function-tested in a 50 µL reaction containing 50 mM Tris-HCl (pH 8.0), 2 mM MgCl 2, 100 mM NaCl, 20 μg rRNA and varying amounts of enzyme.Contaminating activity assays: Terminator Exonuclease is free of detectable contaminating endonuclease and non-5′-phosphate-dependent nuclease activities.4. General considerationsIntegrity of the RNA sample: The success of a Terminator Exonuclease reaction is strongly influenced by the quality of the total RNA sample used in the reaction. Therefore, it is important to confirm the purity and integrity of the RNA sample prior to beginning the reaction.RNA sample: The RNA sample to be treated with Terminator Exonuclease should be dissolved in RNase-free water prior to treatment.IMPORTANT: Do not dissolve the RNA sample in TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) or any other EDTA-containing buffer. EDTA concentrations as low as 1 mM will inhibit Terminator Exonuclease activity.Maintaining an RNase-free environment: Ribonuclease contamination is a significant concern for those working with RNA. The ubiquitous RNase A is a highly stable and active ribonuclease that can contaminate any lab environment and is present on human skin.Creating an RNase-free work environment and maintaining RNase-free solutions is critical for performing successful reactions. Therefore, we strongly recommend that the user:1) Autoclave all tubes and pipette tips that will be used in the reactions.2) Always wear gloves when handling samples containing RNA. Change gloves frequently especiallyafter touching potential sources of RNase contamination such as door knobs, pens, pencils and human skin.3) Always wear gloves when handling reaction components. Do not pick up the enzyme or buffer withan ungloved hand.4) Keep all components tightly sealed when not in use. Keep all tubes containing RNA tightly sealedduring the incubation steps.Buffer/protocol choice: Terminator 5′-Phosphate-Dependent Exonuclease is a processive 5′→3′ exonuclease that digests RNA that has a 5′ monophosphate. It does not digest RNA that has a5′-triphosphate, 5′-cap or 5′-hydroxyl group. However, on rare occasions, we have observed a secondary, non-5′-monophosphate-specific activity on 5′-triphosphate and 5′-hydroxyl RNAs. Because of this activity, two different 10X Reaction Buffers are provided with this kit. The Terminator 10X Reaction Buffer A and its affiliated protocol constitute the standard buffer and protocol. They have been optimised for maximum 5′-monophosphate RNA digestion (e.g., rRNA digestion). The Terminator 10X Reaction Buffer B and its affiliated protocol are provided as an alternate method that should only be used in the rare event that a specific RNA of interest is being degraded by a secondary non-5′-monophosphate-specific activity. Note, however, that less rRNA will be degraded with this buffer and protocol.NOTE: For best digestion, total input RNA must be of high quality (RNA Integrity Number of at least 8 when evaluated on an Agilent Bioanalyzer Model 2100).NOTE: Using the two different buffers in a 50:50 ratio will not produce a reaction condition with the benefits of each individual buffer and protocol.5. Standard procedureIMPORTANT: Two different reaction buffers and protocols are included with Terminator Exonuclease. Be sure to read Buffer/Protocol Choice (section above) to determine which protocol is appropriate for your intended use. Use one or the other. Do not mix buffers.This protocol uses the Terminator 10X Reaction Buffer A and has been optimised for maximum5′-monophosphate rRNA digestion (e.g., rRNA digestion). Buffer B may be used for very low-mass RNA samples.The RNA sample to be treated should be dissolved in RNase-free water. Do not dissolve the RNA sample in TE buffer.1. Gently mix and briefly centrifuge the Terminator 10X Reaction Buffer A prior to use.2. In a sterile (RNase-free) 0.2 mL or 0.5 mL tube, combine the following reaction components on ice:x μL RNase-free water2 µL Terminator 10X Reaction Buffer A0.5 µL RiboGuard RNase Inhibitory µL Total RNA Sample (200 ng-10 μg)1 μL Terminator Exonuclease (1 unit)20 μL Total reaction volume3. Incubate the reaction at 30 °C for 60 minutes in a thermocycler (with heated lid) or Water bath.4. Terminate the reaction by one of the two methods described in Section 6, Step 4.6. Alternative procedureIMPORTANT: Two different reaction buffers and protocols are included with Terminator Exonuclease. Be sure to read Buffer/Protocol Choice (section above) to determine which protocol is appropriate for your intended use.This protocol uses the Terminator 10X Reaction Buffer B and should only be used in the rare event that a specific RNA of interest is being degraded by a secondary non-5′-monophosphate-specific activity of the enzyme.NOTE: Less rRNA will be degraded with this buffer and protocol.The RNA sample to be treated should be dissolved in RNase-free Water. Do not dissolve the RNA sample in TE buffer.1. Gently mix and briefly centrifuge the Terminator 10X Reaction Buffer B prior to use.2. In a sterile (RNase-free) 0.2-mL or 0.5-mL tube, combine the following reaction components on ice:x μL RNase-free water2 µL Terminator 10X Reaction Buffer B0.5 µL RiboGuard RNase Inhibitory Total RNA Sample (1-2.5 μg)1 μL Terminator Exonuclease (1 unit)20 μL Total reaction volume3. Incubate the reaction at 42 °C for 30 minutes in a thermocycler (With heated lid) or Water bath.4. Terminate the reaction by one of the two methods described below:4a. Terminate the reaction by adding 1 µL of 100 mM EDTA (pH 8.0).Place the reaction on ice. Note that the enriched mRNA sample will now contain 5 mM EDTA (as well as tRNA, nucleotides and other small RNAs). It may be used directly for applications in which the EDTA will not be inhibitory. However, the high concentration of EDTA may interfere with some subsequent uses of the mRNA, such as RT-PCR. Therefore, it may be necessary to remove the excess EDTA by LiCl precipitation (see beloW), ethanol precipitation or use of anRNA purification column.4b. Terminate the reaction by phenol extraction and ethanol precipitation.1. Add RNase-free water to the reaction to a total volume of 200 µL. Extract once withbuffer-saturated phenol.2. Transfer the aqueous phase to a new RNase-free tube.3. Add 0.1 volume of 3 M sodium acetate and 2.5 volumes of ethanol to the aqueous phaseand mix thoroughly.4. Incubate on ice or at -20 °C for 30 minutes.5. Pellet the RNA by centrifugation in a microcentrifuge for 30 minutes at full speed at 4 °C.6. Carefully remove and discard the supernatant. Do not disturb the RNA pellet which containsthe mRNA.7. Wash the RNA pellet with 70% ethanol to remove residual salt. Resuspend the RNA pellet inRNase-free water or TE buffer.5. (Optional) Purify the enriched mRNA. If desired, the enriched mRNA can be purified from excessEDTA, tRNA, 5S rRNA and other small RNA species, by LiCl precipitation or by using a commercial RNA purification column.Lithium chloride precipitation should be performed only if the original reaction contains >2 μg of total RNA. If the amount of total RNA in the reaction was less than 2 μg, purify the mRNA by phenol extraction and ethanol precipitation as described above or using a commercial RNA purification column.Lithium chloride selectively precipitates large RNA such as mRNA, while small RNA such as tRNA1. Add 1 volume of 5 M LiCl solution to the sample and mix well.2. Incubate on ice or at -20 °C for 30 minutes.3. Pellet the RNA in a microcentrifuge for 30 minutes at full speed at 4 °C.4. Carefully remove and discard the supernatant Which contains the tRNA, other small RNAs andnucleotides. Do not disturb the RNA pellet which contains the mRNA.5. Wash the RNA pellet with 70% ethanol to remove residual salt.6. Resuspend the RNA pellet in RNase-free Water or TE buffer.7. (Optional) Analyse the effectiveness of the Terminator reaction by denaturing agarose gelelectrophoresis or using an Agilent 2100 Bioanalyzer. When using either method, it is important to run an untreated RNA sample (~250 ng of total RNA) alongside the treated sample. Theabsence of 18S and 28S rRNA in the post-treatment sample indicates a successful reaction.7. Further supportIf you require any further support, please do not hesitate to contact our Technical Support Team:************************.All trademarks and registered trademarks mentioned herein are the property of their respective owners. All other trademarks and registered trademarks are the property of LGC and its subsidiaries. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording or any retrieval system, without the written permission of the copyright holder. © LGC Limited, 2023. All rights reserved. GEN/894/EK/0223@LGCBiosearch。

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