By: Emily Antonides

Single Nucleotide Polymorphisms (SNPs) on Poor Quality or Low

Concentration DNA samples

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 flourescent TaqMan oligonucleotide probes pairs

Results

Results

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 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. <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/>.