By: Emily Antonides
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Transcript of By: Emily Antonides
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/>.