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Download Alu-TPA PCR Kit (#8) Tech Service Training August ‘99
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The Search for a Jumping Gene: Use PCR to Determine Your Molecular Genotype AdaptedModule by Dan Murray from aon presentation by: based a kit from Stan Hitomi - Monte Vista High School, Danville, Bio-Rad Laboratories, Inc. CA. Kirk Brown - Tracy High School, Tracy, CA. Outline • • • • • • What is PCR? PCR Mechanism Transposable Elements PV92 PCR Experimental Protocol Expected Results What is PCR? What is PCR? • PCR stands for Polymerase Chain Reaction • The technique was invented by Kary Mullis in 1983. Awarded the Nobel Prize in Chemistry in 1993. What is PCR? • DNA replication gone crazy in a tube! • Only a small region of the DNA actually gets replicated What’s in a PCR Reaction? • Source DNA – contains target DNA region to be copied • Primers – short single stranded DNAs complementary to ends of target DNA region • Nucleotides – dATP, dCTP, dGTP, dTTP; the building blocks of DNA strands • Taq DNA polymerase – temperature resistant enzyme which builds DNA strands PCR Mechanism How does PCR work? • Mix PCR reaction reagents • Cycle through various temperatures • 94oC – Melting • ~55oC – Annealing • 72oC – Extension • Repeat Melting: 94ºC Heat separates strands 5’ 3’ 3’ 5’ 94oC 5’ 3’ 3’ 5’ Annealing: ~55ºC Primers bind template 5’ 3’ 3’ 5’ ~55oC 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ Extension: 72ºC Taq polymerase builds strands 3’ 5’ 3’ 5’ 3’ 3’ 5’ 5’ 72oC 5’ 3’ 3’ 5’ 3’ 3’ 5’ 5’ Desired-length PCR product made starting in third cycle Cycle 1 5’ 3’ 5’ 5’ 3’ 5’ Cycle 2 3’ 5’ 3’ Cycle 3 3’ 3’ 5’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ Transposable Elements What are Transposable Elements? • Segments of DNA which have the ability to move to or be copied to other regions of the genome Replicate are thought Element • Generally, they of as “Selfish DNA”; they usually neither benefit nor harm their host • Discovered by Barbara McClintock (1940s-50s) while working with maize Donor molecule Recipient molecule Alu Elements • Alu elements are a type of transposable element • Alu elements are 300bp in length • Found only in primates (including humans) • Approx. 500,000 Alu copies per haploid genome, representing about 5% of the genome! • Named for the Alu I restriction site within the element Alu at PV92 • PV92 is a locus on human chromosome 16 • An Alu insertion occurred there within the last 1,000,000 years • Possible Genotypes at PV92: +/+ +/- -/- • Used in population genetics, paternity analysis, and forensics • Not associated with any phenotype PV92 PCR The Target DNA Chromosome 16 PV92 Locus Alu Element Some Got It, Some Don’t PV92 with Alu PV92 without Alu PCR PCR Experimental Protocol The Procedures in a Nutshell • Isolate DNA from cheek cells • Build PCR reaction • Do PCR in thermal cycler • Analyze using agarose electrophoresis Isolation of Cheek Cell DNA •Collect cheek cells and place in InstaGene Matrix •Matrix binds cellular Mg2+ •Agitate •Disperses cells in Matrix Isolation of Cheek Cell DNA 56°C Loosens connective tissue and inactivates nucleases 100°C Ruptures cell membranes and denatures proteins Isolation of Cheek Cell DNA Gets rid of InstaGene Matrix Target DNA now ready! Assemble PCR Reaction Add PCR Master Mix containing: •Nucleotides •Primers •Reaction buffer •Electrophoresis dyes •Taq polymerase PCR reaction now ready to go! Amplify DNA with PCR Controls will show the three possible outcomes Load, Run, and Stain Gel Stain gels and analyze. Expected Results PV92 Amplification Specifics Primer binding sites No Alu: Region to be amplified With Alu: Now assume an Alu element has inserted at that locus Alutoelement Region be amplified PV92 Amplification Specifics No Alu: 641bp Region to be amplified With Alu: 941bp 300bp Region to be amplified Actual Results +/+ -/- +/- 941 bp 641 bp