Transcript Slide 1
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 to complete DNA replication in a cell? http://www.johnkyrk.com/DNAreplication.html 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) 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 View the molecular structure of DNA (see the 5’ to 3’ structure): http://207.207.4.198/pub/flash/24/menu.swf Denaturing Template DNA Heat causes DNA strands to separate 5’ 3’ 3’ 5’ Denaturation of DNA at 94oC 5’ 3’ 3’ 5’ 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’ 3’ 5’ 5’ Repeat denaturing, annealing, and extending 40 cycles The exact-length target product is made in the third cycle 5’ Cycle 1 Cycle 2 Cycle 3 3’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ View the PCR reaction at the Dolan DNA Learning center: http://www.dnalc.org/ddnalc/resources/shockwave/pcranwh ole.html The target sequence PV92 Alu insertion: ~300 bp Found on chromosome 16 5’ Alu Amplified Region 3’ PV92 Alu insertion A member of Alu repeat family Repetitive sequences make up almost half of the human genome, Alu repeats are just one kind of repeat Human-specific Alu insertion Intron: Found in a non-coding region of your DNA NOT diagnostic for any disease or disorder 5’ Alu Amplified Region 3’ Alu repeat family Classified as SINEs (Short Interspersed Repetitive Element) Named for the Alu I restriction site within the element Approx. 1 million Alu copies per haploid genome, representing about 11% of the genome: role in genetic architecture and genetic disorders Approximately one insertion every 200 new births Subfamily members by diagnostic substitutions or sequence mutations accumulated over time: JSY This Alu insert is of subfamily Ya5 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 Amplified in primate genomes through RNA-dependent mechanism, retroposition Homologous Recombination Parent DNA duplexes align at similar sequences and new DNA is formed by breaking and joining of homologous sequences. RecA protein (ATPpowered) mediated through catalyzing strand exchange Key role in DNA repair Retroposition Retroposon 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 RNA polymerase Retroposon mRNA Reverse transcriptase Retroposon DNA Original Retroposon Integration Protein Duplicated retroposon 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 Evolutionary Significance of PV92 Alu Inserts 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 PCR Results The PV92 Alu is dimorphic so there are two possible PCR products: No insertion: 641 bp 641 bp Alu insertion: 941 bp 641 bp 941 bp 300 bp Alu insert What are the possible Genotypes? Chromosome 16 Homologous Chromosomes 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? PV92 Locus Homozygous positive: +/+ Homozygous negative: -/Heterozygous: +/- Actual Alu PCR Results + - +/- 941 bp 641 bp + - +/- PCR Procedures Day 1 Day 2 Day 3 Day 4 Genomic DNA Extraction = Chelex® cation exchange resin; binds cellular MgCl2 56oC loosens connective tissue and inactivates DNases 100oC ruptures cell membranes and denatures proteins InstaGene InstaGene Extraction Cell membrane Nuclear membrane Mg++ Genomic DNA Mg++ Mg++ Heat disrupts membranes Mg++ Mg++ Mg++ InstaGene matrix binds released cellular Mg++ 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 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 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