High-throughput real-time PCR-based genotyping without DNA purification

BMC Research Notes

Nathan R Treff¹  Chaim Jalas²  Jing Su¹  Anastasia Fedick³ 

[1] Reproductive Medicine Associates of New Jersey, Morristown, NJ, USA;Bonei Olam, Center for Rare Jewish Genetic Disorders, 1755 46th St, Brooklyn, NY, 11204, USA;RMA of NJ ATTN, Anastasia Fedick 111 Madison Ave, Suite 100, Morristown, NJ, 07960, USA

关键词: QuantStudio;    Sample-to-SNP;    Blood;    Genotyping;    High-throughput;   

Others  :  1165446
DOI  :  10.1186/1756-0500-5-573

 received in 2012-06-19, accepted in 2012-10-16,  发布年份 2012

【 摘 要 】

Background

While improvements in genotyping technology have allowed for increased throughput and reduced time and expense, protocols remain hindered by the slow upstream steps of isolating, purifying, and normalizing DNA. Various methods exist for genotyping samples directly through blood, without having to purify the DNA first. These procedures were designed to be used on smaller throughput systems, however, and have not yet been tested for use on current high-throughput real-time (q)PCR based genotyping platforms. In this paper, a method of quantitative qPCR-based genotyping on blood without DNA purification was developed using a high-throughput qPCR platform.

Findings

The performances of either DNA purified from blood or the same blood samples without DNA purification were evaluated through qPCR-based genotyping. First, 60 different mutations prevalent in the Ashkenazi Jewish population were genotyped in 12 Ashkenazi Jewish individuals using the QuantStudio™12K Flex Real-Time PCR System. Genotyping directly from blood gave a call rate of 99.21%, and an accuracy of 100%, while the purified DNA gave a call rate of 92.49%, and an accuracy of 99.74%. Although no statistical difference was found for these parameters, an F test comparing the standard deviations of the wild type clusters for the two different methods indicated significantly less variation when genotyping directly from blood instead of after DNA purification. To further establish the ability to perform high-throughput qPCR based genotyping directly from blood, 96 individuals of Ashkenazi Jewish decent were genotyped for the same 60 mutations (5,760 genotypes in 5 hours) and resulted in a call rate of 98.38% and a diagnostic accuracy of 99.77%.

Conclusion

This study shows that accurate qPCR-based high-throughput genotyping can be performed without DNA purification. The direct use of blood may further expedite the entire genotyping process, reduce costs, and avoid tracking errors which can occur during sample DNA purification.

【 授权许可】

   
2012 Fedick et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Wagner FF, Bittner R, Petershofen EK, Doescher A, Müller TH: Cost-efficient sequence-specific priming-polymerase chain reaction screening for blood donors with rare phenotypes. Transfusion 2008, 48(6):1169-1173.
• [2]Guo JR, Schnieder F, Abd-Elsalam KA, Verreet JA: Rapid and efficient extraction of genomic DNA from different phytopathogenic fungi using DNAzol reagent. Biotechnol Lett 2005, 27(1):3-6.
• [3]Kramvis A, Bukofzer S, Kew MC: Comparison of Hepatitis B Virus DNA Extractions from Serum by the QIAamp Blood Kit, GeneReleaser, and the Phenol-Chloroform Method. J Clin Microbiol 1996, 34(11):2731-2733.
• [4]Li Y, Mustapha A: Evaluation of four template preparation methods for polymerase chain reaction-based detection of Salmonella in ground beef and chicken. Lett Appl Microbiol 2002, 35(6):508-512.
• [5]Rose HL, Dewey CA, Ely MS, Willoughby SL, Parsons TM, Cox V, Spencer PM, Weller SA: Comparison of Eight Methods for the Extraction of Bacillus atrophaeus Spore DNA from Eleven Common Interferents and a Common Swab. PLoSOne 2011, 6(7):e22668.
• [6]Fedick A, Su J, Treff NR: Development of TaqMan allelic discrimination based genotyping of large DNA deletions. Genomics 2012, 99:127-131.
• [7]Guillermit H, Fanen P, Ferec C: A 3' splice site consensus sequence mutation in the cystic fibrosis gene. Hum Genet 1990, 85:450-453.
• [8]Kaul R, Gao GP, Aloya M, Balamurugan K, Petrosky A, Michals K, Matalon R: Canavan Disease: Mutations among Jewish and Non-Jewish Patients. Am J Hum Genet 1994, 55:34-41.
• [9]Levran O, Desnick RJ, Schuchman EH: Niemann-Pick type B disease. Identification of a single codon deletion in the acid sphingomyelinase gene and genotype/phenotype correlations in type A and B patients. J Clin Invest 1991, 88(3):806-810.
• [10]Beutler E, Gelbart T, Kuhl W, Sorge J, West C: Identification of the second common Jewish Gaucher disease mutation makes possible population-based screening for the heterozygous state. Proc Natl Acad Sci USA 1991, 88(2):10544-10547.
• [11]Myerowitz R, Costigan FC: The Major Defect in Ashkenazi Jews with Tay-Sachs Disease Is an Insertion in the Gene for the α-Chain of β-Hexosaminidase. J Biol Chem 1988, 263(35):18587-18589.
• [12]Hollegaard MV, Grove J, Thorsen P, Nørgaard-Pedersen B, Hougaard DM: High-Throughput Genotyping on Archived Dried Blood Spot Samples. Genet Test Mol Bioma 2009, 13(2):173-179.
• [13]Treff NR, Su J, Tao X, Northrop LE, Scott RT Jr: Single-cell whole-genome amplification technique impacts the accuracy of SNP microarray-based genotyping and copy number analyses. Mol Hum Reprod 2011, 17:335-343.
• [14]Tzvetkov MV, Meineke I, Sehrt D, Vormfelde SV, Brockmöller J: Amelogenin-based sex identification as a strategy to control the identity of DNA samples in genetic association studies. Pharmacogenomics 2010, 11(3):449-457.
• [15]Mychaleckyj JC, Farber EA, Chmielewski J, Artale J, Light LS, Bowden DW, Hou X, Marcovina SM: Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage. J Transl Med 2011, 9:91. BioMed Central Full Text