PV92 PCR/Informatics Kit Alu insert, PV92 locus, chromosome 16

What can you do with the Chromosome 16 PV92 PCR kit ? Introduce the polymerase chain reaction (PCR) technique Apply PCR to population genetics Directly measure human diversity at the molecular level Compare results to published data online

What is PCR? DNA replication gone crazy in a tube! Makes many copies of a specific target sequence from a small amount of template DNA Affects gene mapping and cloning and DNA sequencing and detection Applications in the detection of specific mutations, criminal investigations, and the human genome

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 Template: do not need to know sequence of desired template; only the sequence of sections on either side Primers: need forward and reverse for double stranded DNA Nucleotides: equal amounts of each are needed to extend the DNA MgCl2: Enzyme cofactor needed for Taq polymerase to function Buffer: maintains proper pH during thermal cycling Taq polymerase: Heat stable polymerase originally found in bacteria in Yellowstone Natn’l park

How does PCR work? Repeat multiple cycles 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’ Denaturation of DNA at 94oC

Annealing Primers Primers anneal at 60oC Primers bind to the template sequence Taq polymerase recognizes double-stranded substrate 5’ 3’ 3’ 5’ Primers anneal at 60oC 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’

Taq polymerase extends….. Taq polymerase extends primer DNA is replicated 5’ 3’ 5’ 3’ 3’ 5’ Extend at 72oC 3’ 5’ 3’ 5’ 5’ 3’ Repeat denaturing, annealing, and extending 40 cycles

The exact-length target product is made in the third cycle 3’ 5’ Cycle 1 3’ 5’ 3’ 5’ Cycle 2 3’ 5’ 3’ 5’ 3’ 5’ Cycle 3 View the PCR reaction at the Dolan DNA Learning center: http://www.dnalc.org/shockwave/pcranwhole.html

The target sequence PV92 Alu insertion Found on chromosome 16 5’ Alu 3’ Amplified Region

PV92 Alu insertion Amplified Region A member of Alu repeat family Human-specific Alu insertion Intron: Found in a non-coding region of your DNA NOT diagnostic for any disease or disorder 5’ 3’ Alu Amplified Region

Introns vs. Exons Introns are non-coding segments of DNA and remain “inside” the nucleus Exons are segments of DNA that code for proteins and they “exit” the nucleus picture, Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cumming

Alu repeats Classified as SINEs (Short Interspersed Repetitive Element) Approx. 500,000 Alu copies per haploid genome, representing about 5% of the genome Named for the Alu I restriction site within the element

Evolutionary Significance of PV92 Alu Inserts Highly conserved Inserted in the last 1,000,000 years Used in population genetics, paternity analysis, and forensics

PCR Results The PV92 Alu is dimorphic so there are two possible PCR products: No insertion: 641 bp 641 bp 941 bp Alu insertion: 941 bp 641 bp 300 bp Alu insert

Actual Alu PCR Results 941 bp 641 bp - +/- + + - +/-

PCR Procedures Day 1 Day 2 Day 3 Add Master Mix containing: Nucleotides Primers Reaction buffer Electrophoresis dyes Taq polymerase PCR Procedures

Genomic DNA Extraction InstaGene = Chelex® cation exchange resin;binds cellular MgCl2 56oC loosens connective tissue and inactivates DNases 100oC ruptures cell membranes and denatures proteins InstaGene Matrix: DNases, which break down DNA, need cations like Mg++ to function. The beads of chelex bind these cations which prevents DNases from breaking down the DNA we wish to extract

InstaGene matrix binds released cellular Mg++ InstaGene Extraction Cell membrane Nuclear membrane Mg++ Genomic DNA Mg++ Mg++ Heat disrupts membranes Mg++ Mg++ InstaGene matrix binds released cellular Mg++ Mg++

Extensions Population Genetics Informatics