连锁关联分工析在疾病研究中的探讨.pptVIP

* * Illumina’s genotyping assay is called the GoldenGate Assay It is a high specificity extension and ligation assay that allows the simultaneous analysis of over 1500 loci in a single reaction and uses the UNIVERSAL Sentrix arrays by incorporating into the reaction products a unique 地址 sequence for each locus being interrogated that is matched to a specific bead type’s illumiCode One of the most important features of the assay is that it uses genomic DNA and not amplified DNA as the genotyping target For each locus being interrogated 3 specific oligos are used 2 allele specific oligos (ASOs) , both of which have a sequence region that is a perfect complement to the genomic region directly adjacent to the target SNP site but that differ in their 3 prime base such that they only match one of the 2 alleles at the site, and a second region that acts as a universal primer site for the subsequent amplification reaction A third locus specific oligo (LSO) hybridizes between 1 to 20 bases downstream of the target SNP site through a complementary sequence. Mention benefit of flexible LSO placement is avoidance of hybridizing over other SNPs in close proximity The LSO also contains two other components, a unique illumiCode sequence that perfectly matches an illumiCode oligo on an array bead and a third universal primer sequence. After oligo hybridization, a polymerase with high specificity for 3’ mismatch is added and only extends the ASO(s) that perfectly match the target sequence at the SNP site. As the polymerase used has no strand displacement or exonuclease activity when it hits the LSO it simply drops off the genomic DNA At this time a DNA Ligase joins the extended sequence to the LSO to from a superstructure that is a template for highly multiplexed PCR One important thing to note is that typically over 1000 loci on the genomic DNA are being interrogated at the same time and as such a population of over 3000 distinct oligos is present in the reaction tube. Can als