Alu insert, PV92 locus, chromosome 16 PV92 PCR Alu insert, PV92 locus, chromosome 16

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

The target sequence… Your DNA!!!! Only Chromosome 16: Out of 23 total In metaphase Human Cheek Cell Your DNA!!!! Only Chromosome 16: Out of 23 total Only the Alu insertion at the PV92 locus: A 600 base pair region out of over 3 billion in your total genome! Key PCR Concepts: Small amount of template DNA Target specific regions of DNA Fast results Chromosome 16 Histones Base Pairs DNA Double Helix

PV92 Alu insert NOT diagnostic for any disease or disorder! A member of Alu repeat family Repetitive sequences make up almost half of the human genome, Alu repeats are just one kind 306 base pair segment of DNA, Classified as a SINE (Short Interspersed Repetitive Element) Named for the Alu I restriction site within the sequence Human-specific Alu insertion Approx. 1 million Alu copies per haploid genome = 11% of the genome: role in genetic architecture and genetic disorders Intron: Found in a non-coding region of your DNA NOT diagnostic for any disease or disorder!

Alu sequence GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA 306 base pairs long: This sequence remains the same, no matter where it is found in the genome

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

The PV92 Alu is dimorphic so there are two possible PCR products: 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

Possible Genotypes? PV92 Locus Each gene locus has a particular form of the gene, or allele What are the possible alleles for the Alu insert at each locus? +, Alu present -, Alu not present What are the possible genotypes for the Alu insert for any given person? Homozygous positive: +/+ Homozygous negative: -/- Heterozygous: +/- Chromosome 16 Homologous Chromosomes PV92 Locus

Forward Reverse 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) Buffer, containing salt Taq polymerase Magnesium chloride (enzyme cofactor) 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

Repeat multiple cycles 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 5’ 3’ 3’ 5’ Denaturation of DNA at 94oC 5’ 3’ 3’ 5’

Primers anneal at 60oC Annealing Primers 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/ddnalc/resources/shockwave/pcranwhole.html

PV92 locus sequence with Alu AACTGGGAAAATTTGAAGAGAAAGTCACACAGATACATTTCAGTAAGGTTGTCTCTGTTACTTGAGGCTTACAAGAAGGAAAGAAGGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAGAAAGAATTCCCTCTCTAAACACACTCTAACACACAGGAGTTGAGAACTCA Where would your primers be if you were looking for this alu insert anywhere in the genome? Where would your primers be if you were looking for only the alu insert at the PV92 locus?

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