chapter 4 Manipulation of purified DNA
生物化学 Chapter 4
Copyright © 2013 Pearson Canada Inc.
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Biochemistry, 4th Edition
Based on the following, which is true regarding the acid/base properties of guanine? A) at physiological pH, guanine will be positively charged B) at physiological pH, guanine will be negatively charged C) at physiological pH, guanine will be neutral D) at physiological pH, guanine will be a very effective buffer against base E) at physiological pH, guanine will be a very effective buffer against acid
4 - 10
Biochemistry, 4th Edition
The Nature of Nucleic Acids
Ultraviolet absorption spectra of ribonucleotides.
This strong absorbance is often used for quantitative determination of nucleic acids because it allows measurement of nucleic acid concentrations at the microgram/mL level by measurement of light absorption at 260 nm.
CHAPTER 4 翻译
the secondary structure
dihydrouracil
pseud ouridin e
4.2.1 tRNA的二级结构
D臂是根据它含有二氢尿嘧啶命名的。 反密码子臂是根据位于套索中央的三联反密码子 命名的。 TψC臂是根据3个核苷酸命名的，其中ψ表示拟尿 嘧啶，是tRNA分子所拥有的不常见核苷酸。 受体臂（acceptor arm）主要由链两端序列碱 基配对形成的杆状结构和3’端末配对的3-4个碱 基所组成，其3’端的最后3个碱基序列永远是 CCA，最后一个碱基的3’或2’自由羟基可以被氨 酰化。
Reaction step: First, the aminoacyltRNA synthetase attaches AMP to the-COOH group of the amino acid utilizing ATP to create an aminoacyl adenylate intermediate(氨酰腺 苷酸中间体). Then, the appropriate tRNA displaces the AMP.
4.密码子与反密码子的相互作用
tRNA反密码子 第1位碱基 A密码子 第3位碱基 I 次黄嘌呤 核苷 U G A C
U, C, A A, G U, C U
G
解释了反密码子中稀有成分（如I）的配对 以及许多氨基酸有2个以上密码子的问题。
密码子与反密码子的摆动配对
U
反密码子Wobble hypothesis
4.2.2 tRNA 的 L型三级结构
4.2.3 tRNA的功能
1. 专一性识别和结合氨基酸，形成氨酰tRNA。氨基酸必须与一种接合体结合，才能被
带到RNA模板的恰当位置上正确合成蛋白质(形成 AA-tRNA复合物)。
过程步骤详解中英文对照
实验四蛋白质印迹分析【实验目的】了解蛋白质印迹法的基本原理及其操作和应用。
【实验原理】蛋白质印迹法又称为免疫印迹法，这是一种可以检测固定在固相载体上蛋白质的免疫化学技术方法。
待测蛋白既可以是粗提物也可以经过一定的分离和纯化, 另外这项技术的应用需要利用待测蛋白的单克隆或多克隆抗体进行识别。
如图所示，可溶性抗原，也就是待测蛋白首先要根据其性质，如分子量，分子大小，电荷以及其等电点等采用不同的电泳方法进行分离；通过电流将凝胶中的蛋白质转移到聚偏二氟乙烯膜上；利用抗体(一抗) 与抗原发生特异性结合的原理，以抗体作为探针钓取目的蛋白。
值得注意的是在加入一抗前应首先加入非特异性蛋白，如牛血清白蛋白对膜进行“封阻” 而防止抗体与膜的非特异性结合。
经电泳分离后的蛋白往往需再利用电泳方法将蛋白质转移到固相载体上, 我们把这个过程称为电泳印迹。
常用的两种电转移方法分别为:1.半干法: 凝胶和固相载体被夹在用缓冲溶液浸湿的滤纸之间,通电时间为10 分钟~30 分钟。
2. 湿法：凝胶和固相载体夹心浸放在转移缓冲溶液中，转移时间可从45 分钟延长到过夜进行。
由于湿法的使用弹性更大并且没有明显浪费更多的时间和原料，因此我们在这里只描述湿法的基本操作过程。
对于目的蛋白的识别需要采用能够识别一抗的第二抗体。
该抗体往往是购买的成品，已经被结合或标记了特定的试剂，如辣根过氧化物酶。
这种标记是利用辣根过氧化物酶所催化的一个比色反应，该反应的产物有特定的颜色且固定在固相载体上，容易鉴别。
因此可通过对二抗的识别而识别一抗，进而判断出目标蛋白所在的位置。
其他的识别系统包括碱性磷酸酶系统和125I 标记系统。
【实验材料】1. 实验器材SDS/PAGE实验相关材料；电转移装置；供电设备；PVDF膜 ( Millipore Immobion-P #IPVH 000 10 ); Whatman 3MM纸；其他工具：镊子、海绵垫、剪子、手套、小塑料或玻璃容器、浅盘。
医学人文Chapter4基础医学
医学人文Chapter 4基础医学您的姓名：［填空题］*1.It is a ____ that the French eat so much rich food and yet have a relatively low rate of heart disease.（）［单选题］*A.dilemmaB.paradox（正确答案）C.pinnacleD.affluence2.After a long struggle against cancer, her condition began to _______ further.（）［单选题］*A.deteriorate（正确答案）B.defineC.detectD.decline3.She was ____ with her daughter for behaving so awkwardly.（）［单选题］*A.glimpsedB.elicitedC.rumpledD.irritated（正确答案）4.Hewas the only person who understood all the ______ details of the agreement.（）［单选题］*A.erraticB.harshC.arcane（正确答案）D.intractable5.The state of the economy has _____ all the other issues as the major item of current public concern.（）［单选题］*A.suppliedB.supportedC.supplanted（正确答案）D.submitted6.In future the authorities will not tolerate any ______ of the safety regulations.（）［单选题］*A.breach（正确答案）B.banishC.branchD.brink7.He had an ____ childhood because it was during the war and there were no luxuries then.（）［单选题］*A.abundant8.austere（正确答案）9.aortic10.audible8.Hosting the Olympic Games added to our country’ in s ternational _______ .（）［单选题］A.privilegeB.perfectionC.perceptionD.prestige（正确答案）9.The poison produced by the spider’ sk s in is so _______ t hat it will paralyze a bird or a monkey immediately.（）［单选题］*A.lethal（正确答案）B.legalC.staggeringD.brooding10.She seems completely unaware of the contradictions in her professed point of view. （）［单选题］*A.inheritedB.inherent（正确答案）C.intravenousD.intimate11. A significant proportion of what medical students are studying at college will be out ofwithin a few years.（）［单选题］*A.dataB.availabilityC.date（正确答案）D.accessibility12.This is not all your fault; the responsibility was ______ mine.（）［单选题］*B.in relation toC.in regard toD.in part（正确答案）13.The law will apply equally to men and women except in the _______ of maternity leave. （）［单选题］*A.studyB.case（正确答案）C.situationD.example14.Technology is advancing at such a staggering pace that it is difficult to keep _________ of it.（）［单选题］*A.abreast（正确答案）B.aboveC.asideD.across15.He was far too _____ w ith his own marital difficulties to give any thought to his friend’ pr s oblems.（）［单选题］*A.precautiousB.preoccupied（正确答案）C.preliminaryD.prescribed16.In the ____ of her research she discovered some important facts about the flu pandemic.（）［单选题］*B. causeC.caseD. caution17.His decision to move overseas has a lot to do _______ his financial problems.（）［单选题］*A.onB.ofC.aboutD.with（正确答案）18.What we did in the previous case is no guide to what we ought to do in this, as the two are not .（）［单选题］*A.on the contraryB.on purposeC.on a par（正确答案）D.on the whole19.____ the patients r’esentment and complaints, the U.S. medical profession is tryinghard to get back to a principle as old asHippocrates.（）［单选题］*A.In generalB.Instead ofC.For all（正确答案）D.For instance20.Economic growth must not be pursued at the _______ of environmental pollution.（）［单选题］*B.extinctionC.expectationD.expense（正确答案）21.Each visitor expects not only medical care but comfort, sympathy, relief, reassurance and ___.（）［单选题］*A.solace（正确答案）B.pinnacleC.paradoxD.quantum22.It is a situation that both ______ the patient and worries the medical profession.（）［单选题］*A.deterioratesB.elicitsC.irritates（正确答案）D.supplants23.Because the patient is increasingly _______ about medicine, thanks to better reportage of medical advances, he is also likely to demand more of his doctor.（）［单选题］*atoseB.preoccupiedC.ingrainedD.sophisticated（正确答案）24.One result of all this change is a growing _________ in the practice of medicine that has created a breach in the traditional doctor-patient relationship.（）［单选题］*A.impersonality（正确答案）B.predecessorC.affluenceD.absurdity25.Doctors can point out that their fees have risen just about ________ with the general cost of living in recent years.（）［单选题］*A.on a par（正确答案）B.in partC.for allD.as a whole26.Today's patient, who is ________ enough to realize his doctor's limitations, is willing to extend that trust.（）［单选题］*A.staggeringB.austereC.obsoleteD.sophisticated（正确答案）27.The patient’co s ncern, his uneasiness, about doctors and doctoring is deeply _________ . （）［单选题］*A.lethalB.self-anointedC.ingrained（正确答案）D.arcane28.Over the ages, doctors have compounded both the awe and the anxiety by acting as apriesthood whose rites and methods were beyond the understanding of any outsider.（）［单选题］*A.lethalB.self-anointed（正确答案）C.ingrainedD.arcane29.The commonest complaint is about the doctor’ in s creasing _______________ .（）［单选题］*A.absurdityB.priesthoodC.inaccessibility（正确答案）D.panjandrum30.In return he wants some understanding and sympathy, the vital _________ t hat nowadays are too often missing.（）［单选题］*A.prestigeB.ingredients（正确答案）C.absurdityD.quantum。
分子生物学英文版Chapter4
CHAPTER4 Translation4.1 Outline of Translation 4.2 Genetic Code4.3 tRNA and Anticodon4.4 Ribosome4.5 Protein Synthesis4.6 Posttranslational Events4.1 Outline of TranslationFrom mRNA to protein4.2 Genetic CodeProtein: 20 different amino acidsmRNA: 4 different basesA single base as a codon: 4 codonsPairs of bases as codons: 16 codonsTriplets of bases as codens: 64 codonsThe genetic code is the correspondence between base sequences in DNA (or RNA) and amino acids in protein.A codon is a triplet of nucleotides that represents an amino acid or a start/termination signal of translation.The genetic code is triplet终止密码子：UAA(赭石, ochre); UAG(琥珀, amber),UGA(蛋白石, opal)起始密码子: AUG (有些为GUG)A reading frame is one of the three possible ways of reading a nucleotide sequence. Each reading frame divides the sequence into a series of successive triplets.Open reading frameAn open reading frame (ORF)is a sequence of DNA consisting of triplets that can be translated into amino acids starting with an initiation codon and ending with a termination codon.There are three possible ways of translating any nucleotide sequence into protein, depending on the starting point. For example:For the sequence ACGACGACGACGACGACG……the three possible reading frames are:(AUG) ACG ACG ACG ACG ACG ACG ACG….. (AUG) CGA CGA CGA CGA CGA CGA CGA….. (AUG) GAC GAC GAC GAC GAC GAC GAC…..遗传密码的特点1. 遗传密码的简并性简并性(degeneracy):指一个氨基酸有一个以上的密码子为其编码。
翻译——精选推荐
细胞分子生物学文章第十卷（2005），711-719 pl2005.7.15寄出200510.6收到脂质体：一项先进制造技术的概述新西兰，北帕默斯顿，专用邮袋11222，梅西大学Riddet中心，M.REZA MOZAFARI摘要：近几十年来，脂质体作为生物膜的理想模型，也是药物、诊断、疫苗、营养物和其他生物活性剂的有效载体，引起了广泛关注。
在不同背景下研究者们对脂质体学领域的文献报道广泛地不断地增加，这表明这一领域引人入胜。
自从大约40年前脂质体被介绍到科学界，许多技术和方法在或大或小的脂质体制造规模上得到发展。
这篇文章将在大体上提供脂质体制备方法优缺点的概览，特别强调在我们实验室开发的加热法，作为一种脂质囊泡快速生产的模式技术。
关键词：载体系统，加热法，脂质囊泡，脂质体学，制造技术引言脂质体科学技术是一个正在飞速发展的科学，举几个例子，它用于诸如药物递送，化妆品，生物膜的结构和功能，探索生命起源等领域。
这是由于脂质体有一些有利的特性，例如，它不仅能包含水溶性药物也能包含脂溶性药物，在体内识别特定靶向位点，在流动性、大小、电荷、层数的方面具有多样性。
脂质体作为生物膜模型的应用限于在实验室中研究，它们在生物活性剂的包载和递送的成功应用不仅取决于脂质体载体可以达到预期目的的优越性的示范，还取决于技术和经济可行性的规划。
对于递送应用，脂质体配方应该具有高包封率，窄粒度分布，持久稳定性和理想的释放特性（根据预期的应用）。
这些要求制备方法有产生脂质体的可能性，且脂质体可采用多种成分分子，例如：脂质/磷脂可提高脂质体稳定性。
除了上述特性，对于蛋白质、核酸之类敏感的分子/化合物的递送，脂质体也应该能保护复合制剂，防止其退化。
尽管在脂质体上进行了大量的研究开发工作，但只有少数脂质体产品已被批准为人类使用至今。
这也许有许多原因：一些脂质体配方的毒性，分子和化合物在脂质体中的低包封，脂质体载体的不稳定性，脂质载体的不稳定性，特别是大尺度的脂质体生产成本高。
生物化学《DNA的生物合成》课件
三、DNA复制反应呈半不连续性
The Reaction of DNA Replication Exhibits Semidiscontinuous Replication
参与DNA复制的DNA聚合酶，必须以一段具有3'端自由羟基（3'-OH）的RNA作为引物(primer) ，
才能开始聚合子代DNA链。 RNA引物的大小，在原核生物中通常为50~100个 核苷酸，而在真核生物中约为10个核苷酸。RNA引 物的碱基顺序，与其模板DNA的碱基顺序相配对。
pol Ⅰ为具有三种酶活性 的单一肽链的大分子蛋白 质，可被特异的蛋白酶水 解为两个片段，其中的大 片段保留了两种酶活性, 即5'→3'聚合酶和3'→5' 外切酶活性，通常被称为 Klenow fragment。
Klenow片段的分子结构
➢ DNA-pol Ⅰ （109kD）
功能： 对复制中的错误进行校读，对复制和修复
3´ T C G A A G பைடு நூலகம் C C T A G C G A C 5´
• 3 5外切酶活性 能辨认错配的碱基对，并将其水解。
• 5 3外切酶活性 能切除突变的 DNA片段。
（一）原核生物至少有三种DNA聚合酶
在原核生物中发现的DNA聚合酶至少有三种， 分别命名为DNA聚合酶Ⅰ（pol Ⅰ），DNA聚 合酶Ⅱ（pol Ⅱ），DNA聚合酶Ⅲ（pol Ⅲ）， 这三种酶都属于具有多种酶活性的多功能酶。 参与DNA复制的主要是pol Ⅲ和pol Ⅰ。
DNA的复制、转录和翻译过程就构成了遗传学的 中心法则。
在RNA病毒中，其遗传信息贮存在RNA分子中。 因此，在这些生物体中，遗传信息的流向是RNA 通过复制，将遗传信息由亲代传递给子代，通过 反转录将遗传信息传递给DNA，再由DNA通过转 录和翻译传递给蛋白质，这种遗传信息的流向就 称为反中心法则。
国际疾病分类编码第十版
国际疾病分类编码第十版Chapter I 第一章Certain infectious and parasitic diseases(A00-B99) 特定感染症及寄生虫疾病（A00-B99）Chapter II第二章Neoplasms(C00-D48) 肿瘤（C00-D48）Chapter III第三章Diseases of the blood and blood forming organs and certain disorders involving the immune mechanism(D50-D89)血液和造血器官及涉及免疫机转的疾患（D50-D89）Chapter IV第四章Endocrine,nutritional and metabolic diseases(E00-E90) 内分泌营养及新陈代谢疾病（E00-E90）Chapter V 第五章Mental and behavioural disorders(F00-F99) 精神与行为障碍（F00-F99）Chapter VI第六章Diseases of the nervous system(G00-G99) 神经系统疾病（G00-G99）Chapter VII第七章DISEASES OF THE EYE AND ADNEXA(H00-H59) 眼睛和附属器官的疾病（H00-H59）Chapter VIII第八章Diseases of the ear and mastoid process(H60-H95) 耳及乳突之疾病（H60-H95）Chapter IX第九章DISEASES OF THE CIRCULATORY SYSTEM(I00-I99) 循环系统疾病（I00-I99）Chapter X 第十章DISEASES OF THE RESPIRATORY SYSTEM(J00-J99) 呼吸系统疾病（J00-J99）Chapter XI第十一章DISEASES OF THE DIGESTIVE SYSTEM(K00-K93) 消化系统疾病（K00-K93）Chapter XII第十二章DISEASES OF THE SKIN AND SUBCUTANEOUS TISSUE(L00-L99) 皮肤及皮下组织疾病（L00-L99）Chapter DISEASES OF THE MUSCULOSSKELETAL SYSTEM AND CONNECTIVEXIII第十三章TISSUE(M00-M99)骨骼肌肉系统及结缔组织之疾病（M00-M99）Chapter XIV第十四章DISEASES OF THE GENITOURINARY SYSTEM(N00-N99) 泌尿生殖器官疾病（N00-N99）Chapter XV第十五章RREGNANCY,DHILDBIRTH AND PUERPERIUM(O00-O99) 妊娠生产及产褥期Chapter XVI第十六章CERTAIN CONDITIONS ORIGINATING IN THE PERINATAL PERIOD(P00-P96)围产期病况（P00-P96）Chapter XVII第十七章CONGENITAL MALFORMATIONS,DEFORMATIONS AND CHROMOSOMAL ABNORMALITIES(Q00-Q99)先天畸形变形及染色体异常（Q00-Q99）Chapter XVIII 第十八章SYMPTOMS,SIGNS AND ABNORMAL CLINICAL AND LABORATORY FINDINGS,NOT ELSEWHERE CLASSIFIED(R00-R99)症状症後与他处未归类之临床及实验室检查异常所见（R00-R99）Chapter XIX第十九章Injury,poisoning and certain other consequences of external causes(S00-T98)外伤中毒和其他外因所造成的特定影响（S00-T98）Chapter XX第二十章External causes of morbidity and martality(V01-Y98) 外伤中毒和其他外因所造成的特定影响（S00-T98）Chapter XXI第二十一章FACTORS INFLUENCING HEALTH STATUS AND CONTACT WITH HEALTH SERVICES(Z00-Z99)影响健康状况及使用医疗服务的因素（Z00-Z99）文- 汉语汉字编辑词条文，wen，从玄从爻。
Gene Manipulation
DNA Ligase
Enzymes that catalyze the formation of a phosphodiester bond between adjacent 3’-OH and 5’-P termini in DNA.
Back
Vector
A self-replicating DNA molecule that transfers a DNA segment between host cells.
❖ Genomic DNA library ❖ Chromosome specific library ❖ cDNA library
Nucleic Acid Probe
A labelled segment of DNA (RNA) that is able, after a DNA hybridization reaction, to detect a specific DNA sequence in a mixture of sequences.
95℃ 72℃ 55℃
Polymerase Chain Reaction
It can be thought of as a molecular photocopier.
Polymerase Chain Reaction
☺ Advantages
❖ Speed ❖ Sensitive ❖ Stable
Polymerase Chain Reaction
❖ Denaturing step: 94~95℃ ❖ Annealing step: 45~60℃ ❖ Extension step: 72℃
Optimal temperature for Taq polymerase to attach to and extend the new strand of DNA
蛋白质的生物合成省名师优质课赛课获奖课件市赛课一等奖课件
Section 2 The Proce需要处理旳问题： 1. 原核生物和真核生物中，多肽链旳生物合成
涉及哪些主要旳环节？ 2. 什么是核糖体循环？核糖体循环主要由哪些
反应过程所构成？ 3. 多肽链合成时，延长一种氨基酸残基需要消
耗多少分子ATP？
factor，EF）。
EF-Tu bound with ribosome
原核生物中存在3种延长因子（EF-TU，EF-TS， EF-G），真核生物中存在2种（EF1，EF2）。 EF旳作用主要是促使氨基酰tRNA进入核蛋白旳 受位；并可增进移位过程，即具有转位酶活性， 可催化核糖体向mRNA 3’-端移动一种密码子旳 距离，使下一种密码子定位于A位。
摆动配对现象示意图
U
二、核糖体是多肽链生物合成旳场合
Ribosome is the place for peptide biosynthesis
核糖体（又称核蛋白体），是多肽链合成旳场合， 是由多种rRNA与蛋白质组装形成旳复合体。
核糖体旳构成
大肠杆菌核糖体旳空间构 造为一椭圆球体，其30S 亚基呈哑铃状，50S亚基 带有三角，中间凹陷形成 空穴，将30S小亚基抱住， 两亚基旳结合面为多肽链 生物合成旳场合。
（一）原核生物多肽链合成旳起始
涉及下列几种环节： ➢ 核糖体大、小亚基分离； ➢ mRNA在小亚基定位结合； ➢ 起始氨基酰-tRNA旳结合； ➢ 核糖体大亚基结合。
5. 摆动性（wobble）： 转运氨基酸旳tRNA旳反密码需要经过碱基互 补与mRNA上旳遗传密码反平行配对结合。 但反密码与密码之间经常不严格遵守碱基配 对规律，称为摆动配对。
密码子与反密码子旳摆动配对
tRNA反密码子 第1位碱基
同源重组pcr方法
同源重组pcr方法英文回答：The method of homologous recombination PCR (polymerase chain reaction) is a powerful technique used in molecular biology to introduce specific mutations or modifications into a DNA sequence. This technique combines the principles of PCR amplification and homologous recombination to facilitate the precise manipulation of DNA.To perform homologous recombination PCR, several components are required. First, a DNA template containing the target sequence is needed. This can be genomic DNA, plasmid DNA, or any other source of DNA that contains the desired sequence. Next, two primers are designed to flank the target sequence. These primers should have homology to the template DNA, as well as additional sequences that allow for the introduction of mutations or modifications.The PCR reaction is set up by combining the DNAtemplate, primers, nucleotides, and a DNA polymerase enzyme. The reaction mixture is subjected to a series oftemperature cycles, which allows for the denaturation ofthe DNA template, annealing of the primers, and extensionof the DNA strands by the polymerase enzyme. This resultsin the amplification of the target sequence, with the primers incorporating the desired mutations or modifications.After the PCR amplification, the product is typically purified and used for subsequent experiments. This can include cloning the modified DNA into a vector for further analysis or expression, or using the PCR product directlyfor functional studies.Homologous recombination PCR has numerous applicationsin molecular biology research. It can be used to create knock-out or knock-in mutations in genes of interest, allowing for the study of gene function. It can also beused to introduce specific modifications, such as tagging a protein with a fluorescent marker or introducing single nucleotide polymorphisms for disease association studies.Overall, homologous recombination PCR is a versatile and powerful technique that allows for precise manipulation of DNA sequences. It has revolutionized the field of molecular biology and has become an essential tool for researchers worldwide.中文回答：同源重组PCR方法是分子生物学中一种强大的技术，用于在DNA序列中引入特定的突变或修饰。
大肠杆菌表达的基本流程
大肠杆菌表达的基本流程英文回答：The basic process of expressing genes in Escherichia coli, commonly known as E. coli, involves several steps. E. coli is a widely used model organism in molecular biology and genetic engineering due to its ease of cultivation and manipulation.First, a gene of interest is inserted into a vector, which is a DNA molecule that can replicate independently inside the bacterial cell. The vector usually contains a promoter region, which is a DNA sequence that initiates gene expression, and a selection marker, which allows for the identification and selection of bacteria that have successfully taken up the vector.Next, the vector is introduced into E. coli cells through a process called transformation. This can be achieved by treating the cells with calcium chloride orsubjecting them to an electric field, among other methods. The transformed cells are then plated onto agar plates containing a specific antibiotic that kills non-transformed cells. This antibiotic resistance gene is often included in the vector as the selection marker.After incubation, colonies of transformed E. coli cells that have successfully taken up the vector will appear on the agar plates. These colonies can be further confirmed by performing a colony PCR or plasmid isolation to verify the presence of the gene of interest.Once the transformed E. coli colonies are confirmed, a small amount of the culture is inoculated into a liquid growth medium and allowed to grow overnight. This liquid culture serves as the starting point for large-scale production of the protein encoded by the gene of interest.To induce gene expression, a specific inducer molecule is added to the liquid culture. The choice of inducer depends on the promoter used in the vector. For example, the lac operon system is often used, where the inducer isisopropyl β-D-1-thiogalactopyranoside (IPTG). The inducer binds to the lac repressor protein, allowing RNA polymerase to bind to the promoter and initiate transcription of the gene of interest.After induction, the E. coli cells continue to grow and produce the protein of interest. The protein can be intracellular, meaning it remains inside the bacterial cells, or it can be secreted into the culture medium. Different strategies can be employed to optimize protein production, such as controlling the growth conditions, modifying the culture medium, or using specific strains of E. coli.Once the desired amount of protein is produced, thecells are harvested by centrifugation or filtration. The protein of interest can then be purified using various techniques, such as chromatography or affinity purification, to remove impurities and obtain a highly purified protein.中文回答：大肠杆菌（Escherichia coli）基因表达的基本流程包括几个步骤。
exclusive cleavage 解理
In the wild world of molecular biology, exclusive cleavage is like the secret agent of DNA cutting! It's all about slicing up DNA at a precise spot, and the superheroes behind this operation are restriction enzymes. These enzymes have a knack for recognizing specific sequences and then going in for the cut –talk about precision teamwork! Without exclusive cleavage, we wouldn't be able to pull off cool tricks like gene cloning, genetic engineering, or DNA sequencing. So, in the DNA world, exclusive cleavage is kind of a big deal!在分子生物学的野生世界中，排他性分裂就像DNA切割的秘密特工！这完全是为了在精确的位置切除DNA，而这个操作背后的超级英雄是限制酶。
这些酶具有识别特定序列的诀窍，然后进入切口——谈论精准团队合作！没有独家的分裂，我们就无法发挥出很酷的技巧，比如基因克隆，基因工程，或者DNA测序。
在DNA世界中，排他性分裂是件大事！The secret to getting that perfectly exclusive cleavage? It's all about picking the right restriction enzyme, baby! These bad boys are like DNA snipers, homing in on specific sequences called recognition sites and cutting that DNA right at the mark. It's like precision surgery for your genetic material. And here's the kicker - by choosing an enzyme that recognizes a one-of-a-kind sequence in your DNA, you're guaranteeing that it'll only slice and dice where you want it to. Talk about a DNA makeover! This level of specificity is key for scientists to do their DNA manipulation magic in the lab. So, remember, when ites to exclusive cleavage, it's all about that enzyme power!得到那个绝无仅有的分裂的秘密？这都是关于选择正确的限制酶，宝贝！这些坏男孩就像DNA狙击手，跟踪特定的序列，叫做识别地点，并切割DNA 右在标记。
PureSpeed 梯度洗脱技术白皮书说明书
W h i t eP ape r workflow.Pipette tip column chromatographic separations with Rainin ™ XLS pipette and industry standard resins1–12 samples processed in parallel in as little as 15 minutes Superior concentration and purity of recovered protein Gentle on proteins – superior protein activityColorTrak ™ Guide workflow for rapid and error free work PureSpeed Protein Tips are the most efficient and cost effective way to purify recombinant proteins and antibodies. Purification methods are downloaded or written into the E4 XLS pipette using Rainin’s standard E4 XLS software inter-face, and PureSpeed tips containing resin bed are placed on the pipette. Affin-ity purification can be performed with a number of different types of resins.2P r o t e i n P u r i f i c a t i o nThe PureSpeed tip’s unique design allows bi-directional flow of the sample through the tip (see arrow in Figure 1). The resin bed located at the end of the pipette tip contains standard affinity resins, and the back and forth flow of sample through the resin bed maximizes use of the capture functional groups within the resin bed. Capture equilibrium reactions are pushed to completion which, compared with a straight flow through interaction, increases capture amount of the target protein and the reproducibility of the capture. The back and forth buffer flow also increases the wash efficiency of removing non specific bound impurities off the bed. The dead volume of the resin bed is minimized by using screen frits to contain the resin hav-ing extremely low surface area, thickness and pore volume. The design of the screens sur-rounding the resin allows elution volumes as low as 2 to 3 times the resin bed volume, e.g. for a 5 µL resin bed an elution volume as low as 10– 15 µL can be used. For a given amount of purified protein, the concentration of the eluted product will increase as the volume of solu-tion decreases. Thus, because PureSpeed Protein Tips can be eluted with a very low elution volume, very high concentrations of proteins can be recovered.Figure 1. Schematic of the PureSpeed Tip3Figure 2. Heart CutSuperior concentration and purity of recovered proteinPureSpeed tips employ a Virtual Heart-Cut™ elution process to produce the highest possibleconcentration and purity of product with the benefit of parallel processing of up to 12 samples ata time. See Figure 2.In conventional affinity chromatography, the center of the eluted peak (heart cut of the peak) iscaptured to produce the highest concentrated product at the highest possible purity.The leading and trailing edges of the chromatography peak are discarded to remove impurities,leaving the very pure peak center. Collecting at the peak center (heart cut) has the additionalbenefit of producing the highest possible concentration of recovered protein, although this willdepend on how much sample was loaded onto the column.Heart cut collection is an effective technology but can be tedious and very expensive to operatein parallel, when using traditional tools and techniques.4P r o t e i n P u r i f i c a t i o nMaximizing resin capacity The first step of the purification process is loading to full capacity the affinity functional groups on the resin, which is embedded in the PureSpeed tip. This is accomplished by using enough sample and back and forth flow of sample through the tip so that even slow interact-ing proteins, proteins that are slowly captured by the resin, have adequate contact time to be taken up. As many sample aliquots as desired can be loaded onto the tip to ensure that the resin bed is maximally loaded. In the case of limited sample, small resin beds can be used. It seems counter intuitive, but using a smaller resin bed will produce higher concentrations of recovered proteins. Efficient purification High purity is achieved in the next step. Thin, low dead volume and low surface area column frits allow complete washing of the resin bed with a very high wash volume to resin volume ratio (up to 50 times) to thoroughly remove non specific bound contaminants. The frits are hydrophilic and have as much as 100 times less surface area compared with conventional frits so there is less surface area for contaminants to bind. High wash volume ratios and low column surface area allow the non specific bound contaminants to be very effectively re-moved from the resin bed prior to elution. This high efficiency washing provides the highest purity protein.Low volume elution for high concentration proteins Finally, fine control of the elution volume allows very small and complete elution of the puri-fied protein producing the highest possible concentrations. Because the tip is so highly loaded and the elution volume can be controlled, the Virtual Heart-Cut elution process pro-duces a product similar to collecting the heart of a chromatography peak. The elution process is performed with 2–3 bed volumes of solutions producing protein concentrations that are 5-10 times higher than competing technologies. For 5 µL bed tips, elution volumes as low as 10 µL are possible while 80 µL tips can be eluted with as little as 160–240 µL. Additional protein can be recovered with additional ing conventional gravity and spin columns, the approach to obtaining acceptable perfor-mance in a small column format is to use as much affinity resin as seems practical. Unfortunately, this conventional approach does not maximize the potential of the affinity resin to capture protein and consequently, larger volumes of resin are needed to efficiently capture the required protein. PureSpeed tips obtain superior results by using small resin bed volumes — as small as possible — so that the resin bed capacity is loaded up with as much protein as possible.The following set of experiments show how and why superior concentrations and superiorpurity of proteins are recovered with PureSpeed tips compared to spin columns. These dataexplain when and why a researcher would use a 200 µL tip and when they would use the 20µL resin bed vs. the 5 µL resin bed. A standard mastermix of 100 µL or 500 uL of 0.5 mg/mLhIgG was purified on the PureSpeed Protein Tips and as a comparison, with a 200 µL bedspin column. Sample processing on the commercial spin column was performed accordingto manufacturer’s instructions. The flow through (all liquid material at the end of each pro-cess step) and recovered protein from the tips and columns were measured by HPLC and gel electrophoresis.L 2 3 4 5 6 7Lane 1: Protein LadderLane 2: E.coli lysate spiked with hIgGLane 3: Flow throughLane 4: 100 µL lysate purified on Spin column with 200 µL ProALane 5: 100 µL lysate purified on PureSpeed tips with 5 µL ProALane 6: 500 µL lysate purified on Spin column with 200 µL ProALane 7: 500 µL lysate purified on PureSpeed tips with 20 µL ProAFigure 3. NuPAGE® gel showing E.coli lysate spiked with 0.5 mg/mL hIgG and purified using PureSpeed tips and spincolumn.The experiments with the 1000 PureSpeed tips show a similar result (Table 1). Using a 500µL sample with 0.5 mg/mL hIgG, PureSpeed tip offers a comparable amount of antibodyyield despite having a bed size that is 10 times smaller than the spin column. Again, the re-producibility of the recovery from PureSpeed tips is still superior to spin columns.The concentration recovered from the 20 µL tip is dramatically improved over the spin col-umn while the mass recovery is fairly equivalent (Figure 3).In many cases, the recovered protein from a spin column is too dilute to assay or to be usedfor downstream processing. In these cases, the researcher must concentrate the recoveredprotein. Many researchers use a membrane spin column such as a Microcon to attempt toaccomplish this. However, a 30 minute spin usually results in a further loss of 50 percent ofthe sample. The PureSpeed tip eliminates this extra step of concentration, speeding the totalprocess time while yielding a higher concentration of protein that has had minimalmanipulation.56P r o t e i n P u r i f i c a t i o n The purity of proteins recovered with the PureSpeed tip is superior compared to spin col-umns. The placement of the screens, the material used and the wash process are all strin-gent, yet gentle on the protein. Small bed volumes and low dead volume screens lead to more efficient wash of non specific bound materials. These results show that non specific contaminants can be washed from PureSpeed Protein Tips much more effectively than from the spin column. The data shown in Table 1 indicate that the concentration and purity of the protein recovered from the 20 µL PureSpeed tip is better than the spin column and the highest concentration of recovered protein is obtained from the smallest resin bed, the 5 µL PureSpeed tip. The repro-ducibility of the spin columns is variable (poorer) compared with the PureSpeed tip (Table 1). This may be due to inefficient mixing (although performed according to specification) of the 100 µL sample with the 200 µL of resin in the spin column. The back and forth flow motion of the PureSpeed tip pushes the interaction equilibrium to completion so the recovery is more reproducible from tip to tip.The missing high molecular weight bands in the elution lane for the spin column (lane 4) in-dicate that the amount loaded is too dilute to detect by the Coomassie staining technique. For this gel, 4 µL of sample was loaded into each well. Since the concentrations of the protein re-covered from the spin columns are low, the target IgG bands are weak and cannot be seen.In comparison, the bands of the target IgG recovered from the PureSpeed tips are very strong. Table 1. HPLC data showing final protein concentration and total yield from PureSpeed tips and Spin columnsThe recovery process from the spin column requires a 400 µL elution step compared with the60 µL elution for the 200 / 20 µL tips and 15 µL elution for the 200 / 5 µL tips. This results indilution of the recovered material of between 6 and 8 fold for the spin columns but an in-crease in concentration of final product of between 2.7 and 2.9 for the PureSpeed tips. In thecase of the 200 / 5 µL tip, the resin is loaded to such a high density that the protein eluted offof the column is recovered at an even higher concentration. In all cases the samples are pureas visualized by gel.GuidelinesThe following are general guidelines to choose when and how to use the various PureSpeedtip body sizes and bed sizes.a) Use the 200 / 5 µL bed tip for samples less than 200 µL. This column produces ultrahighconcentrations e.g. protein complex reconstitution assays, crystallography screening etc.b) When yield is important and samples volumes are less than 200 µL, use the 200 / 20 µLbed tip. The 200 tips are used, generally, when the researcher has a lot of samples thatrequire small volume purification. Examples of use of these tips include routine analysis,expression screening and optimization, protein-protein interaction analysis, and obtainingprotein reagents for proteomics work.c) Use the 1000 / 20 µL bed tips for sample volumes larger than 200 µL. These tips can beused to obtain the highest concentrations with good recovery. For example, researchersworking on protein complexes wishing to reconstitute the complex in vitro in a controlledmanner with recombinant protein components require high concentrations of pure protein.The Ribosome 30S subunit, for example, is composed of rRNA and over 30 proteins. Re-constitution studies have been going on since 1960, but until now it has always beenslow, tedious work.d) W hen yield is important as well as concentration, use the 1000 / 80 µL bed tips. Forsamples larger than 200 µL.7For more information /rainin Rainin Instrument, LLC7500 Edgewater Drive. Box 2160Oakland, CA 94621-0060a METTLER TOLEDO CompanySubject to technical changes© 1/2012 Rainin Instrument, LLCPrinted in USAMarCom Oakland WP-501 Rev B NoteIon pairing reverse phase HPLC with 254 nm detection was used to measure the protein concentration of the various samples. The HPLC is sensitive to below 70 ng hIgG. This method makes quantification more reliable and sensitive compared to the UV method using a NanoDrop where the signal measured is many times greater than the background.PureSpeed and ColorTrak are trademarks of Rainin Instrument, LLC. Virtual Heart-Cut and Capture Purify Enrich are trademarks of PhyNexus, Inc. NuPAGE is a registered trademark of Life Technologies Corporation. NanoDrop is a registered trademark of Thermo Fisher Scientific, Inc.。
孟德尔式遗传
2.
F2有两种性状表现类型的植株，一种表现为显性性状， 另种表现为隐性性状；并且表现显性性状的植株数与 隐性性状个体数之比接近3:1。
隐性性状在F1中并没有消失，只是被掩盖了，在F2代显性性 状和隐性性状都会表现出来，这就是性状分离(character
segregation)现象。
性状分离现象的解释
14.杂交(cross):
是指不同遗传型的个体进行有性交配。
15.回交(backcross)：杂交产生的子一代个体再与其亲本进
行交配的方式。
16.测交(testcross)：即测验杂交，是指把被测验的个体与
隐性纯合的个体杂交。
第二节 孟德尔第二定律及其遗传分析
一、孟德尔实验及其分析（两对性状的遗传分析） 圆粒、黄色 × 皱粒、绿色 ↓ 圆粒、黄色 ↓（自交） 圆粒、黄色 圆粒、绿色 皱粒、黄色 皱粒、绿色 108 101 32
(二)、分离律的应用
基因型和表现型的概念是建立在单位性状上， 所以当我们谈到生物个体的基因型或表现型时， 往往都是针对所研究的一个或几个单位性状而
言，而不考虑其它性状和基因的差异。
(二)、分离律的应用——
生物性状的遗传方式
针对某一性状或某一突变性状，如何确定该性
状的遗传方式：
质量性状或数量性状 质量性状 核遗传 细胞核遗传 控制 一对或几对基因 细胞质遗传或细胞
现甜粒玉米果穗上结有非甜粒的子实，而非甜粒玉米
果穗上找不到甜粒的子实。这一对相对性状中哪一个 为显性性状？怎样验证你的解释？
（三）孟德尔分析的关键性名词概念
1．基因(gene)：
孟德尔在遗传分析中所提出的遗传因子，如决定豌 豆种子的圆形 (R)和皱缩(r) 等。这些因子用现代的术语来说就是 基因。
F4DNAreplication
directions?
What mechanisms ensure that DNA replicates once per cell division?
What enzymes take part in DNA synthesis?
No 5` to 3` exonuclease activity
End view
Side view
DNA synthesis on the leading and lagging strands. Events at the replication fork are coordinated by a single DNA polymerase III dimer, in an integrated complex with DnaB helicase. This figure shows the replication process already underway (parts (a) through (e) are discussed in the text). The lagging strand is looped so that DNA synthesis proceeds steadily on both the leading and lagging strand templates at the same time. Red arrows indicate the 3·end of the two new strands and the direction of DNA synthesis. Black arrows show the direction of movement of the parent DNA through the complex. An Okazaki fragment is being synthesized on the lagging strand.
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2
1 The range of DNA manipulative of enzymes
• Depending on the type of reaction
① Nucleases are enzymes that cut, shorten, or degrade
nucleic acid molecules.
• Viscoty : larger DNA, more viscous than smaller ones RE cleavage result in a decrease in viscoty • Size: gel electrophoresis agarose: 0.5%---4% polyacrylamide: (polyacrylamide: Bis- acrylamide/19:1--39:1 30%/40%)
• Nucleases degrade DNA molecules by breaking the phosphodiester bonds • Exonucleases remove nucleotides one at a time from the end of a DNA molecule. • Endonucleases are able to break internal phosphodiester bonds within a DNA molecule.
• invitrogen
• Takara
10
• The
central
feature
of
type
II
restriction
endonucleases is that each enzyme has a specific recognition sequence at which it cuts a DNA molecule. • A particular enzyme cleaves DNA at the recognition
• A homopolymer is simply a polymer in which all the subunits are the same. e.g poly(dG), poly(dA) • Tailing involves using the enzyme terminal deoxynucleotidyl transferase to add a series of nucleotides onto the 3′-OH termini of a doublestranded DNA molecule.
34
3.3 Putting sticky ends onto a blunt-ended molecule Linker
• • • • • Synthesized in the test tube Short , Double-stranded DNA Known nucleotide sequence Blunt-ended Contains a restriction site
4
5
• The main distinction between different exonucleases lies in the number of strands that are degraded when a double-stranded molecule is attacked.
Both strands of a dsDNA
One strand of a dsDNA
6
Endonucleases
S1 nuclease only cleaves single strands
DNase1 cuts both singleand double stranded molecules
② Ligases join nucleic acid molecules together. ③ Modifying enzymes remove or add chemical groups. ④ Polymerases make copies of molecules.
3
2 Nucleases
ones have been isolated and more than 300 are available for use in the laboratory. • Type Ⅰ, Ⅱ, Ⅲ
9
• NEB (New england biolabs), USA The Leader in Enzyme Technology • Fermentas (MBI) --Thermo • Promega • Roche
14
15
Isocaudarner (同尾酶)
16
(4) The frequency of recognition sequences in a DNA molecule
• A tetranucleotide sequence (e.g., GATC) should occur once every 44 = 256 nucleotides, and a hexanucleotide (e.g., GGATCC) once every 46 = 4096 nucleotides.
• 1970, H Smith et al
• 1978, Nobel prize
8
• These degradative enzymes are called restriction
endonucleases and are synthesized by many,
perhaps all, species of bacteria: over 4000 diffeounts, at a high concentration • No recognize site of RE used in linker in the bluntend molecule
36
• Adaptors
37
38
39
• Producing sticky ends by homopolymer tailing
27
(7) Estimation of the sizes of DNA molecules
• DNA marker
• D = a − b(log M)
• D: the distance moved • M: the molecular mass • a and b are constants that depend on the electrophoresis conditions.
19
10×buffer
1μl
H2O
RE DNA total
6.5μl
0.5μl 2μl 10 μl
20
• Incubation temperature: most 37 ℃ • “Kill” the enzyme: phenol, EDTA, heat…
21
(6) Analyzing the result of restriction endonuclease cleavage
18
(5) Performing a restriction digest in the lab
• 10×buffer ------ 1×buffer Tris-HCl: pH 7.4 Bgl II 10×buffer MgCl: all type II RE require Mg2+ in order to function • NaCl: ionic strength (different enzymes vary in their requirements for ionic strength) Dithiothreitol (DTT,二硫苏糖醇): stabilizes the enzyme and prevents its inactivation.
Restriction endonuclease: specific recognition sites
7
Restriction enzyme
(1) The discovery of restriction endonucleases
• 1950s, Restriction-modification system
28
(8) Restriction map/physical map
29
30
3 DNA ligase-joining DNA molecules
• The two reactions catalyzed by DNA ligase. • (a) Repair of a discontinuity—a missing phosphodiester bond in one strand of a double stranded molecule. • (b) Joining two molecules together.
12%, 15%...
loading buffer
22
23
24
Ethidium bromide (EtBr) Non-mutagenic dyes
25
Silver nitrate (AgNO3)--PAGE
26
Orthogonal field alternation gel electrophoresis (OFAGE) larger DNA >50 kb
• 4n (assume that the nucleotides are ordered in a random fashion and that the four different nucleotides are present in equal proportions GC%=50%)
17
• Hexanucleotide: 49000/4096≈12 • In fact: