How many SNPs are there in Humans today?Human Mutation rate is ~2.5 x 10-8mutations/site/gen~150 mutations/diploidgenome/generation6.8 billion people in the world=1,020,000,000,000 mutations in the=1,020,000,000,000 mutations in theworld today.With 3 billion nucleotides = eachnucleotide in the world today is mutated340 times.
What can SNPs tell you?Linkage DisequilibriumRecombinationAssociation StudiesDemographic eventsDemographic events
Linkage DisequilibriumThe non-random association betweenalleles in a population.No LD2 SNPs 4 HaplotypesHigh LD2 SNPs 2 Haplotyps
Association studiesAn association between a genetic variant and aphenotype.Two groups: Disease Group Control (random pop)Look for a variant which is in high freq in your diseasegroup in respect to the control.Cystic Fibrosis was the first successful associationstudy, based on RFLPs. (Kerem et al Science 1989)SNP at Promoter (-169C- susceptible/T nonsusceptible) region of FCRL3 for Rheumatoid arthritisin Japanese population (Kochi et al., Nat Genet. 2005Jun; 37(6):652.)
SNP typing TechniquesHybridizationMethodsEnzyme basedMethodsOther methodsbased on physicalProperties of DNA1. DASH2. Molecular beacons1. RFLP2. Invader Assay1. SSCP2. TGGESome commercialtechniques3. Primer Extension4. Oligonucleotide ligation assay5. Taqman assay2. TGGE1. Gene chip array- Affymetrix2. Bead array- Illumina3. Pyrosequencing – Illumina
DASHPCR is performed using one of the primers contains a 5’biotin.Resulting products immobilized by a streptavidin-coatedmicrotiter plate.The non-biotynylated strand is removed by NaOHsolution.Hybridized with an oligonucleotide probe, (hybridizationbuffer containing a fluorescent double-strand-specificbuffer containing a fluorescent double-strand-specificintercalating dye).The sample is heated slowly from room temperature toabove denaturing temperature while continuallymonitoring fluorescence.By plotting the negative derivative (slope of thefluorescence Vs temp.), denaturation points are clearlyseen as peaks. Peak temperature values can be usedfor final allele determination.
Primer extension (SPE- Mass analysis)Tag-It approach (Tm Bioscience corp, Canada)
SSCPGenomic DNAPCR amplificationDenatured by heat/Denatured by heat/formaldehydeRun on non-denaturingelectrophoresis gel
Pros & consPROSRapidTechnically simpleInexpensiveUses commonly available equipmentDetects 60-95% of mutations in short DNA strandsDetects 60-95% of mutations in short DNA strandsCONSNeed to sequence DNA to identify specific mutationssDNA conformation is highly dependent on temperature andit is not generally apparent what the ideal temperature is.Sensitivity drops when sequences longer than 400 bp areused (Costabile et al. 2006).
CGGCCGGGGCCAATNormal DNATarget DNADenaturing followed by annealingHomoduplexes Homoduplexes HeteroduplexesCG CA TTHomoduplexes Homoduplexes HeteroduplexesTGE TGETGE
Genechip array- AffymetrixAllele-specific probesconsisting of 25-meroligonucleotides aresynthesized in an orderedsynthesized in an orderedfashion to form a probe array
PyrosequencingMix genomic DNA with PCR reagents & thermocylePurify biotinylated PCR products using Streptavidin-SepharoseAnneal extension primer to single stranded biotinylated PCRproductPyrosequencing reactionThis methodology is good forsmall numbers of SNPssmall numbers of SNPs(less than 15)and on smallsample sizes (1-1000)