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PV92 PCR

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

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PV92 PCR

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

  2. 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

  3. The target sequence… Chromosome 16: In metaphase Human Cheek Cell Chromosome 16 • 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 Histones Base Pairs DNA Double Helix

  4. PV92 Alu insert • 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 Alucopies 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!

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

  6. 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

  7. PCR Results • The PV92Aluis dimorphic so there are two possible PCR products: • 641 bp • 941 bp No insertion: 641 bp Alu insertion: 941 bp 641 bp 300 bp Alu insert

  8. Chromosome 16 Homologous Chromosomes PV92 Locus Possible Genotypes? • 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: +/-

  9. 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 • Magnesiumchloride (enzyme cofactor)

  10. 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

  11. 3’ 3’ 5’ 5’ 5’ 5’ 3’ 3’ Denaturing Template DNA Heat causes DNA strands to separate Denaturation of DNA at 94oC

  12. 5’ 3’ 3’ 5’ 5’ 3’ Annealing Primers • Primers bind to the template sequence • Taq polymerase recognizes double-stranded substrate 3’ 5’ Primers anneal at 60oC 5’ 3’ 3’ 5’

  13. 3’ 5’ 5’ 3’ 5’ 3’ Taq polymerase extends….. • Taq polymerase extends primer • DNA is replicated 5’ 3’ Extend at 72oC 5’ 3’ 5’ 3’ Repeat denaturing, annealing, and extending 40 cycles

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

  15. PV92 locus sequence withAlu • 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?

  16. - +/- + 941 bp 641 bp + - +/- Actual Alu PCR Results

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