1. By: Emily Antonides Single Nucleotide Polymorphisms (SNPs) on Poor Quality or Low Concentration DNA samples

2. 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/

3. Methods Used for SNP Detection  Real-Time PCR  Microarrays  Pyrosequencing  Fluorescence homogenous assays  Nucleotide extension  Cleavage  Mass spectrometry  Oligonucleotide ligation

4. 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

5. 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

6. 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

7. 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

8. Promoter region and first part of the coding sequence of the MBL2 gene

10. 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

11. 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 flourescent TaqMan oligonucleotide probes pairs

13. Results

15. 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

16. References  Catsburg, Arnold, Wil C. Van Der Zwet, 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..  "SNPs: Variations on a Theme." NCBI: A Science Primer. N.p., 20 Sept. 2007. Web. Nov. 2012..  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..