PV92PCRpresentation.ppt.pptVIP

* * * * * * * * * * * * Chromosome 16:PV92 PCR What is PCR? DNA replication gone crazy in a tube! Makes many copies of target sequence from template DNA What is needed for PCR? Template (the DNA you want to amplify for the study) Sequence-specific primers flanking the target sequence Forward Reverse Nucleotides (dATP, dCTP, dGTP, dTTP) Magnesium chloride (enzyme cofactor) Buffer, containing salt Taq polymerase (heat stable) How does PCR work? Heat (94oC) to denature DNA strands Cool (60oC) to anneal primers to template Warm (72oC) to activate Taq polymerase, which extends primers and replicates DNA Repeat multiple cycles Denaturing Template DNA Heat causes DNA strands to separate 3’ 5’ 5’ 3’ Denaturation of DNA at 94oC 5’ 3’ 3’ 5’ Annealing Primers Primers bind to the template sequence Taq polymerase recognizes double-stranded substrate 3’ 5’ 5’ 3’ Primers anneal at 60oC 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ Taq polymerase extends….. 3’ 5’ 3’ 5’ 3’ 5’ Extend at 72oC 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ Taq polymerase extends primer DNA is replicated Repeat denaturing, annealing, and extending 40 cycles The exact-length target product is made in the third cycle 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ 3’ Cycle 1 Cycle 2 Cycle 3 3’ 3’ 3’ 3’ 5’ 5’ 5’ 5’ What are we going to amplify? The genome contains small repetitive DNA elements that have become randomly inserted into the human genome over millions of years Alu repeats Classified as SINEs (Short INterspersed Repetitive Element) About 300 base pairs long repeated Approx. 500,000 times throughout the genome, representing about 5% of the genome Named for the Alu I restriction site within the element Function unknown Useful as a measure of genetic variation, associated with disease or used for DNA typing The target sequence PV92 Alu insertion Found on chromosome 16 5’ 3’ Alu Amplified Region PV92 Alu insertion A member of Alu repeat family Human-specific Alu insertion Found in a non-coding region of your DNA NOT diagnostic fo