Product description P140-C1-0114 HBA-v32vcia

COA Version 01; Issued 01-03-2014Certificate of Analysis SALSA® MLPA® probemix P140 HBACatalogue # Product name P140-025R, P140-50R, P140-100R Probemix P140 HBA C1-0114 25, 50 or 100 reactions. Shipping conditions Shipped on dry ice or with cooling elements. Store between -25 °C and -15 °C. January 2019. This product can be frozen / thawed at least 20 times. Use This product has been developed to determine the DNA copy number 34 sequences in the human HBA1 and HBA2 gene region, as described in Table 1 and 2 of the Product Description. This probemix is designed for use in combination with SALSA MLPA reagent kit EK1 (100 reactions) or EK5 (500 reactions) as described in the MLPA General Protocol. Sufficient distance between peaks, absence of extra or shoulder peaks, complete hybridisation of each individual probe, as tested on Applied Biosystems 3130 and Beckman GeXP sequencers. Variation of each individual probe < 0.10 when tested on 23 different DNA samples of healthy individuals, purified by various extraction methods. Variation of each individual probe meeting reaction-specific criteria when testing a single DNA sample in various experimental conditions. Conditions tested include mimicking pipetting mistakes, evaporation, deviating thermocycler temperatures; presence of salts affecting sample DNA denaturation or impurities affecting polymerase activity in the sample DNA. Reactions without sample DNA resulted in only 5 major peaks shorter than 120 nt: 4 Q fragments at 64-70-76-82 nt and the unused portion of the 19 nt fluorescent PCR primer. No non-specific peaks longer than 120 nt AND with a peak height of more than 25% of the median of the 4 Q fragments observed. Peaks below this 25% threshold are not expected to affect MLPA reactions when sufficient sample DNA is used. PASSQuality Test 1Quality Test 2PASSQuality Test 3PASSQuality Test 4PASSFOR RESEARCH USE ONLY. FOR IN VITRO USE ONLY. MSDS: The MLPA probemix sold by MRC-Holland does not contain dangerous substances, as defined in European Directive 67/548/EC and its amendments, at concentrations requiring the distribution of Material Safety Data Sheets as specified in European Directives 1999/45/EC and 2001/58/EC. Therefore, a Material Safety Data Sheet is not required for this product. If spills occur, clean with water and follow appropriate site procedures. More information: MRC-Holland bv; Willem Schoutenstraat 1 1057 DL, Amsterdam, The Netherlands E-mail Phone info@ (information & technical questions); order@ (orders) +31 88 8657202 (Order information) +31 88 8657203 (Technical support)Page 1 of 2Certificate of Analysis P140 HBACOA Version 01; Issued 01-03-2014Certificate of AnalysisSALSA® MLPA® probemix P140-C1 HBA sample pictures350009 5 .9 7300001 7 0 .3 42 4 8 .7 5 2 02.332 0 6 .6 2 1 27.00 1 4 4 .9 4 1 3 9 .0 4 1 9 5 .3 0 1 8 9 .4 0 1 8 3 .7 2 309.88 2 6 0 .9 3 2 9 0 .4 5 2 7 6 .5 5 2 8 3 .3 7 2 2 5 .2 9 2 9 8 .9 0 3 3 6 .1 3 1 0 5 .1 5 2 6 8 .6 7 3 2 8 .0 9 3 4 4 .0 61 6 3 .5 92500085.08219.052 4 3 .3 2 2 3 8 .3 92 5 5 .4 31 7 7 .3 1 9 0 .7 7 1 5 2 .1 1200001 5 7 .7 72 1 3 .5 03 62.39 4 8 4 .0 5 409.50 3 9 9 .5 4 4 1 8 .6 2 3 90.08 3 7 2 .0 4 3 8 2 .3 4 472.013 5 3 .8 8150004 6 4 .3 5 4 5 5 .6 2 4 4 5 .6 7 4 37.431 0 0 .3 210000S ig n a l50007 8 .8 4 72.61 6 6 .4 2 6 0 .1 1D y e0 50 100 150 200 250 300 Size (nt) 350 400 450 500Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA® MLPA® probemix P140-C1 HBA (lot C1-0114).4500096.20 1 3 3 .1 4400002 4 9 .0 4 170.56 2 0 6 .7 6 202.39 1 8 9 .4 4 1 8 3 .7 8 139.21 1 63.77 1 95.39 2 1 9 .2 4 2 6 1 .2 9 2 55.52 290.58 2 4 3 .6 1 2 83.42 2 3 8 .4 4 2 76.64350008 5 .5 31 2 7 .1 3 1 4 5 .0 7309.91300001 0 5 .3 3 1 00.51 9 0 .9 71 5 2 .2 11 7 7 .3 92 2 5 .4 4 2 6 8 .7 12 9 9 .0 63 3 6 .2 0 3 2 8 .1 23 4 4 .1 7 3 6 2 .5 0 4 8 3 .9 2 4 0 9 .5 2 3 9 9 .6 0 4 1 8 .8 1 3 9 0 .1 6 4 5 5 .5 0 382.33 3 72.06 445.77 4 3 7 .3 8 4 6 4 .3 0 4 7 1 .9 5250001 5 7 .9 5 2 1 3 .6 120000353.891500010000S ig n a l50007 2 .8 5 6 6 . 6 67 9 . 1 1 6 0 .4 9D y e0 50 100 150 200 250 300 Size (nt) 350 400 450 500Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng SD031 binning DNA analysed with SALSA MLPA probemix P140-C1 HBA (lot C1-0114). The location of the mutation-specific probe is indicated. The height of the 105 nt Y-specific probe is similar to that in male samples, indicating the presence of one copy control plasmid / cell. The signal of the 100 nt X chromosome-specific probe is twice as high as in male samples. This lot was certified by MRC-Holland on May 1, 2014. This certificate is a declaration of analysis at the time of the manufacturing process. All assays were run in compliance with manufacturer’s constraints and kit inserts.Certificate of Analysis P140 HBA Page 2 of 2MRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Product Description SALSA® MLPA® probemix P140-C1 HBATo be used with the MLPA General Protocol. Version C1. See the last page for product history. Catalogue numbers: • P140-025R: SALSA® MLPA® probemix P140 HBA, 25 reactions. • P140-050R: SALSA® MLPA® probemix P140 HBA, 50 reactions. • P140-100R: SALSA® MLPA® probemix P140 HBA, 100 reactions. To be used in combination with SALSA MLPA reagent kit EK1 (100 reactions) or EK5 (500 reactions). MLPA reagent kits are available with different fluorescent dyes, e.g. EK1-FAM, EK5-FAM (for Applied Biosystems sequencers) and EK1-Cy5.0, EK5-Cy5.0 (for Beckman sequencers). Intended use: This SALSA MLPA probemix P140 HBA is a research use only (RUO) assay for the detection of copy number changes in the human HBA1 / HBA2 gene region and is not intended to be used as a standalone assay for clinical decisions. Most, but not all, defects in the HBA gene are copy number changes. The majority of defects that are not detected by MLPA are point mutations in the HBA1 and HBA2 genes. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis. Clinical background: The human alpha globin gene cluster spans about 30 kb and is located on chromosome 16p13.3 at a distance of only 0.2 Mb from the p-telomere. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. The two genes differ slightly over the 5' untranslated regions and the introns, while they differ significantly over the 3' untranslated regions. Two alpha chains plus two beta chains constitute HbA, which in normal adult life comprises about 97% of the total hemoglobin. Alpha chains combined with delta chains constitute HbA2, which together with HbF (fetal hemoglobin) makes up the remaining 3% of adult hemoglobin. Most alpha thalassemias result from genomic deletions. In the lethal Hb Bart syndrome, the two HBA1 and the two HBA2 copies are all absent or defect. In HbH disease, one functional HBA copy remains. In α-thallassemia trait, two functional copies remain while in “silent” αthallassemia, three functional copies are present. More information is available on /books/NBK1435/ This MLPA probemix is expected to detect approximately 90% of all HBA mutations in most populations. See below for the publications on probemix P140 HBA. MLPA technique: The principles of the MLPA technique are described in the MLPA General Protocol. P140-C1 probemix content: This SALSA MLPA probemix P140 HBA contains 45 MLPA probes with amplification products between 130 and 481 nt: 34 probes for the HBA gene region (described in table 2) and 11 reference probes that detect sequences outside this region. One of these 45 probes (136 nt) is specific for the presence of the Constant Spring mutation and will thus not generate a signal in the great majority of samples. HS40 is the major regulatory element of the human alpha-globin locus and is located 40 kb upstream of the zeta-globin gene. Two HS40 specific probes are present. Deletions of only the HS40 region are known. One probe detects a sequence telomeric of HS40 and 5 probes detect sequences centromeric of HBQ1. These flanking probes are only included to delineate the extend of large HBA region copy number changes. Three probes (172, 214 and 220 nt) detect sequences that are present in both HBA1 and HBA2. The HBA1 and HBA2 genes are almost identical. For five locations with a very small sequence difference between the two genes we designed pairs of probes: one detecting the HBA1 sequence and the other detecting the HBA2 sequence. Due to their close proximity, it is possible that the HBA2 sequence at this position in some healthy individuals is changed in a HBA1 sequence (and vice versa) due to gene conversion, without any clinical consequences.SALSA® MLPA® Probemix P140 HBA Page 1 of 16MRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Probe pairs that can be affected in this way are the 160 and 165 nt intron 2 probes; the 244 and 250 nt intron 2 probes; the 190 and 391 nt probes; the 226 and 328 nt probes; the 202 and 373 nt probes. When one of the probes of such a probe pair has an ~50% reduced probe signal (0.5 ratio), while the other probe has an ~50% increased probe signal (1.5 ratio), it is likely that this is a benign polymorphism due to sequence exchange between HBA1 and HBA2, rather than a true deletion and duplication (see Table 3). In addition to the 10 possible polymorphisms due to gene conversion of these 5 probe pairs, three other frequent (benign) polymorphisms have been described and are shown in table 3. This probemix contains nine quality control fragments generating an amplification product between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), one chromosome X and one chromosome Y-specific fragment (Table 1). More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol. Performance characteristics: The exact product performance characteristics cannot easily be determined due to the difficulty in obtaining DNA samples containing deletions or duplications for each of the probes. Performance of this product can be compromised by, among others: impurities in the DNA sample, incomplete DNA denaturation, the use of insufficient or too much sample DNA, the use of insufficient or unsuitable reference samples, problems with capillary electrophoresis or a poor data normalisation procedure. The MLPA General Protocol contains technical guidelines and information on data evaluation / normalization. Internal validation of the MLPA technique in your laboratory is essential and should indicate a standard variation of each probe of 0.10 or lower when testing DNA samples of healthy individuals. Limitations of the procedure: − In most populations, around 10% of genetic defects in the HBA gene are small (point) mutations, most of which will not be detected by using SALSA MLPA probemix P140 HBA. − The combination of a deletion and a similarly sized duplication may result in false negative MLPA results as there is no net change in copy number (example 14 in Table 5). − MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect most inversions or translocations. Even when MLPA did not detect any aberrations, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected. − Sequence changes (e.g. SNPs, point mutations, small indels) in the target sequence detected by a probe can cause false positive results. Mutations/SNPs (at least up to 20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA. − Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known to result in very mild, or no disease manifestation (Schwartz et al, 2007). Duplications that include the first or last exon of a gene (e.g. exons 1-2) might in some cases not result in inactivation of that gene copy. − The complete sequence detected by the probes is available on . This product description contains only partial sequences detected by the probes. Required specimens: Purified DNA from human tissues, free from impurities. For more information, see MLPA General Protocol and the website section on DNA sample treatment. RNAse sample treatment (essential for HBA and HBB MLPA probemixes) Since HBA is heavily expressed in red blood cells, an RNAse treatment of samples is essential for wholeblood derived samples (DNA samples purified from white blood cells are less affected). Without RNAse treatment, HBA mRNA can bind to the 172, 214 and 220 nt probes that detect a sequence within the HBA1 and HBA2 exons, thereby reducing the effective concentration of probes. Please note that some automatic DNA purification methods (e.g. Roche Magnapure) do not include an RNAse treatment. The following method can be used to treat samples: Mix 4 µl sample and 1 µl 0.5 mg/ml RNAse A. Incubate 30 minutes at 37 °C. Continue with the 5 minutes 98 °C DNA denaturation step of the MLPA protocol. RNAse A is extremely stable; it can be diluted in TE and stored at -20 °C. We recommend RNAse A from Promega (code A7973; 4 mg/ml solution), diluted 8 fold in TE (1 ml of 4 mg/ml RNAse is sufficient for ~8000 samples). Do not use more than the recommended amount.SALSA® MLPA® Probemix P140 HBA Page 2 of 16MRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Reference samples: Reference DNA samples should be derived from the same tissue and should be extracted using the same method as patient samples. Reference samples should be derived from unrelated individuals who are from families without a history of hereditary predisposition to thalassemia. More information on the use of reference samples can be found in the MLPA General Protocol. Please note that approximately 10% of samples from healthy Caucasian individuals did show reproducible copy number changes for one or more of the HBA region probes in our experiments. This % might be higher in other populations. In many cases, these copy number changes were the polymorphisms described in table 3. Positive controls: Inclusion of a positive control DNA sample in your experiment is useful but not essential. MRC-Holland has no positive control DNA sample available for this product. Binning DNA sample: A binning DNA sample (SD031) is included with each shipment of the P140 HBA probemix. This DNA sample is a mixture of genomic DNA and a plasmid containing the sequence of the Constant Spring mutation and can be used to determine the amplicon length of the probe for this mutation. Data analysis: For data analysis, software with the appropriate lot specific probe sheet is available free of charge on . The analysis method used for P140-C1 is block normalisation. Normalization of results should be performed within one experiment. A more detailed description on how to evaluate runs and how to perform data analysis is given in the MLPA General Protocol. Confirmation of results: Copy number changes detected by only a single probe always require confirmation by another method. An apparent deletion detected by a single probe can be due to a mutation or polymorphism that prevents ligation of the MLPA probe or that destabilises the binding of a probe oligonucleotide to the sample DNA. Sequence analysis should establish whether any mutations or polymorphisms are present in the probe’s target sequence. The finding of a heterozygous mutation or polymorphism would indicate that two different copies of the sequence are present in the sample DNA and that a false positive MLPA result may have been obtained. Copy number changes detected by more than one consecutive probe may still be a false positive result, see below. They should be confirmed by another independent technique such as long range PCR, gap-PCR, qPCR, array CGH or Southern blotting, whenever possible. Deletions / duplications of more than 50 kb in length can often be confirmed by FISH. Incomplete denaturation of the sample DNA, due to the presence of salt in the DNA sample, can result in false positive deletion results showing a decreased probe signal for consecutive probes that are located in a CpG island. The use of an alternative DNA extraction method may resolve such cases. False positive duplication results due to contamination of DNA samples with cDNA, or with PCR amplicons of individual exons, have also been described (Varga et al, 2012). Analysis of an independently collected secondary DNA sample can exclude this kind of contamination artifacts. Interpretation of results: Alpha-thalassemia is usually inherited in an autosomal recessive manner. The expected results for most HBA region specific MLPA probes are allele copy numbers of 0 (Hb Bart, homozygous deletion), 1 (heterozygous deletion), 2 (normal), 3 (heterozygous duplication) and occasionally 4 (homozygous duplication), corresponding to probe ratios of respectively 0, 0.5, 1.0, 1.5 or 2.0. Three probes in this product detect a sequence that is present in both HBA1 as well as HBA2 (4 copies / cell). The expected results for these three probes are allele copy numbers of 0, 1, 2, 3, 4 (normal), 5, 6, 7 or 8, corresponding to probe ratios of resp. 0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 or 2.0. Examples are in tables 3, 4 and 5. When using MLPA for the first time or whenever changing the DNA extraction method or instruments used, internal validation of the MLPA technique using 16 DNA samples from healthy individuals is strongly recommended. This validation experiment should result in a standard variation of all probes below 0.10. Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal also more subtle changes such as those observed in mosaic cases. Analysis of parental samples may be necessary for correct interpretation of complex results, e.g. when one allele contains a deletion while the other allele contains a duplication.SALSA® MLPA® Probemix P140 HBA Page 3 of 16MRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Inactivation of a single HBA gene copy is not expected to be pathogenic (α+-thalassemia). Alphaºthalassemia results from deletion or dysfunction of respectively two alleles. Deletion or dysfunction of three alleles results in HbH disease. All four α-globin alleles are deleted or inactivated in Hb Bart syndrome. Normal copy number variation in healthy individuals is described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. Users should always verify the latest updates of the database and scientific literature when interpreting their findings. HBA mutation database: http://HBA1.lovd.nl and /globin/hbvar/menu.html. We strongly encourage users to deposit positive results in these databases. Recommendations for the nomenclature to describe deletions / duplications of one or more exons can be found on /mutnomen/. Please report copy number changes detected by the reference probes, false positive results due to SNPs and unusual results to MRC-Holland: info@. Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: . It is the responsibility of the user to be aware of the latest scientific knowledge of the application in question before drawing any conclusions from findings made with this product.Publications with SALSA MLPA Probemix P140 HBAChen Y.J. (2013). The application of multiplex ligation-dependent probe amplification technology in diagnosis and prenatal diagnosis of α-thalassemia. Zhonghua Xue Ye Xue Za Zhi.34:591-4. Coelho A. et al. (2010). Novel large deletions in the human α -globin gene cluster: Clarifying the HS40 long-range regulatory role in the native chromosome environment. Blood Cells Mol.Dis. 45:147-53. Colosimo A. et al. (2011). Application of MLPA assay to characterize unsolved alpha-globin gene rearrangements. Blood Cells, Molecules, and Diseases. 46: 139-144. Harteveld CL et al. (2005). Nine unknown rearrangements in 16p13.3 and 11p15.4 causing alphaand beta-thalassemia characterised by high resolution multiplex ligation-dependent probe amplification. J.Med.Genet. 42:922-931. Please note that this SALSA MLPA P140 probemix is different from the probemix used by Harteveld et al. in this publication. Kim SY et al. (2010). Molecular identification of the novel Gγ-β hybrid hemoglobin: Hb Gγ-β Ulsan (Gγ through 13; β from 19). Blood Cells, Molecules, and Diseases. 45:276-9. Liu JZ et al. (2008). Detection of alpha-thalassemia in China by using multiplex ligation-dependent probe amplification. Hemoglobin. 32: 561-571. Nezhat N. et al. (2012). Detection of deletions/duplications in α-globin cluster by multiplex ligation-dependent probe amplification. Genet.Test Mol. Biomarkers 16:684-8. Origo R. et al. (2013). Complexity of the alpha-globin genotypes identified with thalassemia screening in Sardinia. Blood Cells Mol.Dis. epub. Phylipsen M. et al. (2010). Thalassemia in Western Australia: 11 novel deletions characterized by Multiplex Ligation-dependent Probe Amplification. Blood Cells, Molecules, and Diseases 44:146-51. Phylipsen M. et al. (2012). A novel α(0)-thalassemia deletion in a Greek patient with HbH disease and β-thalasemia trait. Eur.J.Haematol.88:356-62. Wei XF. et al. (2011). Molecular characterization of a novel 27.6-kb deletion causing α+ thalassemia in a Chinese family. Ann Hematol. 90:17–22.Additional ReferencesSchouten J.P. et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acid Res.30:e57 (Original description of the MLPA technique). Schwartz M. et al. (2007) Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat.28:205.Varga RE. et al. (2012) MLPA-based evidence for sequence gain: Pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421:799-801.Related SALSA MLPA probemixesP102 HBB Probes for the HBB gene region.Page 4 of 16 SALSA® MLPA® Probemix P140 HBAMRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Table 1. SALSA MLPA P140-C1 HBA probemixReference HBA Cluster 64-70-76-82 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA 88-92-96 D-fragments: Low signal of 88 OR 96 nt fragment indicates incomplete denaturation 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe 00797-L13645 5q31 Generates a signal only on samples 136 § Ж HBA region, probe S0585-0043-L09493 containing the Constant Spring mutation! 142 * Reference probe 07641-L07326 8p23 147 * HBA region, probe 18098-L22522 0.9 kb upstream HBA2 154 HBA region, probe 08499-L23594 0.2 kb downstream HBA1 160 HBA region, probe 08498-L08422 HBA2 intron 2 165 * HBA region, probe 08498-L21607 HBA1 intron 2 172 * « HBA region, probe 15857-L21812 HBA1+HBA2 exon 3 178 HS-40, probe 04799-L04797 HS-40 184 HBA region, probe 04637-L04018 Between HBA2P and HBA1P 190 HBA region, probe 18096-L22520 Between HBA2 and HBA1 196 * Reference probe 05846-L11214 10q26 202 * HBA region, probe 18880-L24428 Between HBA2 and HBA1 208 * Reference probe 11331-L12056 12p13 214 « HBA region, probe 18881-L06288 HBA1+HBA2 exon 1 220 * « HBA region, probe 18099-L22524 HBA1+HBA2 exon 1 226 * HBA region, probe 18093-L22517 Between HBA2 and HBA1 238 * Reference probe 11435-L12163 1q41 244 HBA region, probe 04633-L23748 HBA2 intron 2 250 * HBA region, probe 04633-L23600 HBA1 intron 2 256 HBA region, probe 08494-L08417 Between HBA2 and HBA1 262 * ¬ DECR2 gene, probe 17613-L23601 Centromeric of HBA region 269 Reference probe 03075-L19996 5p15 277 * ¬ LUC7L gene, 15859-L21960 Centromeric of HBA region 283 HBA region, probe 04638-L23602 0.5 kb downstream HBA1 292 HBA region, probe 04624-L04004 Between HBZ and HBZP 300 * Reference probe 03250-L02687 13q14 310 HBA region, probe 04639-L04020 2.4 kb downstream HBA1 328 HBA region, probe 18092-L22516 0.6 kb before HBA2 exon 1 337 * HBA region, probe 14855-L23604 Between HBA2 and HBA1 346 HBA region, probe 04622-L04001 3.5 kb upstream HBZ 355 Reference probe 00547-L00116 11q22 364 HBA region, probe 04926-L23886 9.3 kb upstream HBZ 373 HBA region, probe 18090-L08415 2.5 kb before HBA2 382 HS-40, probe 04800-L04175 HS-40 391 * HBA region, probe 18097-L22521 3.0 kb before HBA2 400 * HBA region, probe 19233-L25313 HBA1 exon 3 409 Reference probe 03272-L02709 3q29 418 * ¬ AXIN 1 gene, probe 17212-L13393 Centromeric of HBA region 436 * Ж HBA region, probe 17214-0457-L20489 445 * ¬ ITFG3 gene, probe 17227-L20554 Centromeric of HBA region 454 * Reference probe 07607-L07292 15q26 463 * ¬ POLR3K gene, probe 19236-L25316 Telomeric of HBA region 472 * ¬ RGS11 gene, probe 18102-L20488 Centromeric of HBA region 481 * Reference probe 15318-L17117 2q33 * New in version C1, lot 0513. « The 172, 214 and 220 nt probes detect sequences that are present in both HBA1 and HBA2. ¬ Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. § The 136 nt probe will only give a signal on samples containing the Constant spring mutation. Ж The 136 and 436 nt probes consist of three oligonucleotides and have two Ligation sites SALSA® MLPA® Probemix P140 HBA Page 5 of 16Length (nt)SALSA MLPA probeChromosomal positionMRC-HollandProduct Description version 32; Issued 06-06-2014MLPA®Table 2. HBA region probes according to chromosomal locationLength SALSA (nt) MLPA probe463 178 382 364 346 436Ж 292 ~ 184 391 + 373 + 147 328 + 220 « 214 « 160 + 244 + 172 « 19236-L25316 04799-L04797 04800-L04175 04926-L23886 04622-L04001 17214-SP0457L20489 04624-L04004 04637-L04018 18097-L22521 18090-L08415 18098-L22522 18092-L22516 18099-L22524 18881-L06288 08498-L08422 04633-L23748 15857-L21812 S0585-SP0043L09493 18096-L22520 18880-L24428 08494-L08417 14855-L23604 18093-L22517 18099-L22524 18881-L06288 08498-L21607 04633-L23600 15857-L21812Gene / exonLigation site GenbankPartial sequence (24 nt adjacent to ligation site)From p-telomere to POLR3K probeDistance to next probe37 kbPOLR3K gene HS-40 HS-40 9.3 kb upstream HBZ 3.5 kb upstream HBZ 0.2 kb before HBZ Between HBZ & HBZP Between HBA2P & HBA1P 3.0 2.5 0.9 0.6 kb kb kb kb before before before before HBA2 HBA2 HBA2 HBA2 exon exon exon exon 1 1 1 1 AY327548.1 61-62 220-221 NG_000006.1 4528-4529 10229-10230 13536-13535 & 13499-13498 R 20361-20362 28168-28169 30717-30716 R 31233-31232 R 32850-32849 R 33103-33102 R 33733-33732 R 33824-33823 R 34247-34248 34311-34312 34385-34384 RCTGCCCAAGCCA-AGGGTGGAGGCA GGTACTGCTGAT-TACAACCTCTGG GGCTGGGGCTCA-AACCAAGGCCCA CGCAGTGCTAGA-AGGGAGTTCCTG TTCCTCTCCTGT-37 nt spanning oligo-AGTCTAGGAGAG GTGGAGTAGGCT-TTGTGGGGAACT AGTGGCCACAAT-TTGGCAGACAGA AGGAAGGGGTGA-GAATGAGAGAAA ATGTCCAGAAGA-AAAGCGGTGACA GATAAACAAACT-TGGCTCTGGGTA CCGGGAAGGAAC-AAACACCAGGAC AAGAGTGCCGGG-CCGCGAGCGCGC CGCGCCGACCTT-ACCCCAGGCGGC GCGCCTTCCTCT-CAGGGCAGAGGA GGGCCTGGGCCG-CACTGACCCTCT GCAGGGGTGAAC-TCGGCGGGGAGG66.4 kb 0.2 kb 30.0 kb 5.7 kb 3.3 kb 6.9 kb 7.8 kb 2.5 kb 0.5 1.6 0.2 0.6 0.1 0.4 0.1 0.1 0.1 kb kb kb kb kb kb kb kb kbHBA1 +HBA2 exon 1 HBA1 +HBA2 exon 1HBA2 intron 2 HBA2 intron 2HBA1 +HBA2 exon 3 Constant Spring mutation2.6 kb before HBA1 exon 1(0.4 kb after HBA2 exon 3)136 § Ж190 + 202 + 256 337 226 + 220 « 214 « 165 + 250 + 172 «34461-34462 & 34489-3449034974-34975 35489-35490 36050-36051 36629-36628 R 36907-36908 37537-37536 R 37628-37627 R 38051-38052 38122-38123 38196-38195 RCCAAATACCGTC-28 nt spanning oligo-TGCCCGCTGGGCTTCTCTCATTCC-CACCCCTTCCTG GTCACTGCTTTC-CTTCTGGACATGG TTCTCTGCCCAA-GGCAGCTTACCC CTGGAGCATTCA-ACCTCCTCTGGG TCCTGGTGTTTA-TTCCTTCCCGGT AAGAGTGCCGGG-CCGCGAGCGCGC CGCGCCGACCTT-ACCCCAGGCGGC GCGCCTTCCTCG-CAGGGCAGAGGA GGCCCTCGGCCC-CACTGACCCTCT GCAGGGGTGAAC-TCGGCGGGGAGG0.5 kb 0.5 kb 0.6 0.6 0.3 0.6 0.1 0.4 0.1 0.1 0.3 kb kb kb kb kb kb kb kb kb2.1 1.5 0.9 0.6kb kb kb kbbefore before before beforeHBA1 HBA1 HBA1 HBA1exon exon exon exon1 1 1 1HBA1 +HBA2 exon 1 HBA1 +HBA2 exon 1HBA1 intron 2 HBA1 intron 2HBA1 +HBA2 exon 3TGGGACACACAT-GGCTAGAACCTC 154 08499-L08423 0.1 kb after HBA1 exon 3 38459-38460 0.3 kb 283 04638-L23602 0.4 kb after HBA1 exon 3 38785-38786 AAGTCCCACTCC-AGCATGGCTGCA 1.9 kb GTTCACTGCCCT-GAAGAAACACCT 310 04639-L04020 2.3 kb after HBA1 exon 3 40637-40638 1.4 kb 400 19233-L25313 HBQ1 exon 3 42066-42067 TGCTCTCTCGAG-GTCAGGACGCGA 25.1 kb 277 15859-L21960 LUC7L gene ATGTTCCAATGA-AACCAGTGGCAC 33.5 kb GCTGTGATACTT-TTGCCTTTGTCA 445 17227-L20554 ITFG3 gene 31.9 kb 472 18102-L20488 RGS11 gene GAACCTGAGGTT-CCGTGGAATATT 16.3 kb GATCATCGGCAA-AGTGGAGAAGGT 418 17212-L13393 AXIN1 gene 119.4 kb CAGAATCGACAT-TCTCATTAACTG 262 17613-L23601 DECR2 gene The two sequence blocks between the grey lines are almost identical. Ж The 136 and 436 nt probes consist of three oligonucleotides and have two Ligation sites R These probes detect the reverse sequence. « The 172 nt 15857-L21812, the 214 nt 18881-L06288 and 220 nt 18099-L22524 probes each detect two sequences: HBA1 + HBA2. Deletion of a single target site results in only 20-25% decrease in signal of these probes. § The 136 nt probe S0585-SP0043-L09493 will only give a signal on samples containing the Constant Spring mutation. The probe consists of three synthetic oligonucleotides in order to make it both HBA2 and CS mutation specific. This probe has been tested with good results on mutation-positive samples by several users. MRCHolland has only tested this probe on a plasmid containing the mutation sequence.SALSA® MLPA® Probemix P140 HBAPage 6 of 16。