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Single Nucleotide Polymorphisms (SNPs) on Poor Quality or Low Concentration DNA samples. By: Emily Antonides. What Are SNPs?. Minor variations in a person’s DNA sequence Variation occurs with a single nucleotide On average occur 1 out of 300 -1,000 nucleotides
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Single Nucleotide Polymorphisms (SNPs) on Poor Quality or Low Concentration DNA samples By: Emily Antonides
What Are SNPs? • Minor variations in a person’s DNA sequence • Variation occurs with a single nucleotide • On average occur 1 out of 300 -1,000 nucleotides • Variations can be harmless or harmful https://www.23andme.com/gen101/snps/
Methods Used for SNP Detection • Real-Time PCR • Microarrays • Pyrosequencing • Fluorescence homogenous assays • Nucleotide extension • Cleavage • Mass spectrometry • Oligonucleotide ligation
Real-Time PCR • Exponential amplification of short DNA sequences • A pair of primers • DNA probes • Taq polymerase • dNTPs • DNA template • Thermocycler 3 Steps in Every Cycle 1.) Denature at ~95°C 2.) Annealing at ~55°C 3.) Elongation at ~72°C
SNP Detection • Whole blood or white blood cell fractions used as sources of DNA • Sometimes sources can contain small amounts of DNA or only fragmented DNA
Experiment • Arnold Casburg • Department of Medical Microbiology and Infection Control at VU University Medical Center in Amsterdam • Evaluated a new approach for genotyping multiple SNPs in one gene on very low amounts of DNA • Added a pre-amplification step
Mannose Binding Lectin 2 (MBL2) Gene • C-type lectin that plays a role in the innate immune response to infections • Three mutations have been found in the structural region of the molecule • Three mutations are found in the promoter region
Promoter region and first part of the coding sequence of the MBL2 gene
Controls • DNA samples were taken from four subjects with known homozygous haplotypes • They were cloned • Used to determine the SNP detection limit of each assay • All samples were amplified using Real-Time PCR
SNP Analysis 1.) Isolate DNA from plasma samples 2.) Elute dried blood from cards 3.) Pre-amplification step on samples with low amounts of DNA 4.) Six different Real-Time PCR allelic discrimination TaqMan assays were performed - Four different primer pairs - Six different flourescentTaqManoligonucleotide probes pairs
Conclusion • With a pre-amplification PCR it is possible to detect SNPs in samples that contain small amounts of DNA • This pre-amplification step prior to Real-Time PCR SNP analysis has significantly improved the reliability SNP detection
References • Catsburg, Arnold, Wil C. Van DerZwet, Servaas A. Morré, Sander Ouburg, Christina M.J.E. Vandenbroucke-Grauls, and Paul H.M. Savelkoul. "Analysis of Multiple Single Nucleotide Polymorphisms (SNP) on DNA Traces from Plasma and Dried Blood Samples." Journal of Immunological Methods 321.1-2 (2007): 135-41. Print. • Livak, K.J., 1999. Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet. Anal. 14, 143. • Mattarucchi, E., Marsoni, M., Binelli, G., Passi, A., Lo Curto, F., Pasquali, F., Porta, G., 2005. Different real time PCR approaches for the fine quantification of SNP's alleles in DNA pools: Assays development, characterization and pre-validation. J. Biochem. Mol. Bio. 38, 555. • "MBL2." Genetics Home Reference. A Service of the U.S. National Library of Medicine, 19 Nov. 2012. Web. Nov. 2012. <http://ghr.nlm.nih.gov/gene/MBL2>. • "SNPs: Variations on a Theme." NCBI: A Science Primer. N.p., 20 Sept. 2007. Web. Nov. 2012. <http://www.ncbi.nlm.nih.gov/About/primer/snps.html>. • Steffensen, R., Thiel, S., Varming, K., Jersild, C., Jensenius, J.C., 2000. Detection of structural gene mutations and promoter polymorphisms in the mannan-binding lectin (MBL) gene by polymerase chain reaction with sequence-specific primers. J. Immunol. Methods. 241, 33. • "What Are SNPs?" Genetic Testing for Health, Disease & Ancestry; DNA Test. 23andMe, 2012. Web. Nov. 2012. <https://www.23andme.com/>.
