1 / 29

PV92 PCR

PV92 PCR. 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.

adair
Download Presentation

PV92 PCR

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

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

  2. 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

  3. 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

  4. 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

  5. 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 • This is a Ya5 Alu insert: Subfamily members by diagnostic substitutions or sequence mutations accumulated over time, JSY • 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!

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

  7. Alu as a Mutation • Amplified in primate genomes through RNA-dependent mechanism, retroposition • Approximately one insertion every 200 new births • What does that mean???? • Contribute up to 0.4% of human genetic diseases • Inserts within genes, 0.1% • Unequal homologous recombination events between Alu repeats, 0.3% • Hypercholesterolemia, α-thalassemia, BRCA1-related breast cancer

  8. Retroposon RNA polymerase Retroposon mRNA Reverse transcriptase Retroposon DNA Integration Protein Original Retroposon Duplicated retroposon Retroposition • Transposition: Movement of gene from one chromosome to another or movement from one site to another; does not require homology • Transposons: mobile genetic elements that enable genes to move between non-similar sites • Retroposition: Creates genetic diversity • Retroposons: Replicate and move to other sites on DNA through an RNA intermediate

  9. Homologous Recombination • Parent DNA duplexes align at similar sequences and new DNA is formed by breaking and joining of homologous sequences. • RecA protein (ATP-powered) mediated through catalyzing strand exchange • Key role in DNA repair

  10. 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

  11. Evolutionary Significance of Alu • Some inserts are recent enough that they remain polymorphic • Highly conserved • Inserted in the last 1,000,000 years • Used in population genetics, paternity analysis, and forensics • Ancestral state known to be absent of Alu insertion • Alu insert reflects a single, unique event in human evolution

  12. 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

  13. 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: +/-

  14. DNA Replication in Eukaryotes http://www.johnkyrk.com/DNAreplication.html

  15. 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)

  16. 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

  17. View the molecular structure of DNA (see the 5’ to 3’ structure): http://207.207.4.198/pub/flash/24/menu.swf

  18. 3’ 3’ 5’ 5’ 5’ 5’ 3’ 3’ Denaturing Template DNA Heat causes DNA strands to separate Denaturation of DNA at 94oC

  19. 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’

  20. 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

  21. 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

  22. 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?

  23. - +/- + 941 bp 641 bp + - +/- Actual Alu PCR Results

  24. PCR Procedures Day 1 Day 2 Day 3 Day 4

  25. Genomic DNA Extraction • InstaGene= Chelex® cation exchange resin; binds cellular MgCl2 • 56oCloosens connective tissue and inactivates DNases • 100oCruptures cell membranes and denatures proteins

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

  27. Extensions • Making DNA probes • Gene expression studies • Detection of viral pathogens or bacterial infections • Genetic diagnoses • Crime scene investigations • Anthropology • Key words: minimal template and FAST

  28. Microbial Diagnostics • RFLP (a): Banding patterns are compared to reference strains • PCR (b): sensitivity allows small sample size and early detection; clinical microbiology diagnostics and microbe infections • DNA sequencing (c): Microbes affecting public health (76 million cases of food-borne disease causing over 300,00 hospitalizations per year in the US

  29. DNA Fingerprinting • MLP’s (20-30 bands) ensure identification: 1 in 30 billion chance of a match between individuals • In the United States, the FBI incorporates 13 sites on average into its profiles. With 26 different bands studied, you'd be incredibly hard pressed to find two unrelated individual with the same DNA profile; the odds of a match in this case are well more than one in a hundred billion. The bottom line is that, unless you have a twin, you're statistically two thousand times more likely to win the Publisher's Clearinghouse sweepstakes (1 in 50,000,000) than to have a DNA profile that matches anyone else. • http://www.dnai.org/d/index.html

More Related