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Polymerase Chain Reaction

Polymerase Chain Reaction. a.k.a. “How’d they get all that DNA from just a little blood?”. What does PCR stand for?. Polymerase Chain Reaction Developed by Kary Mullis – Nobel Prize Received a $20,000 bonus; later sold it to Hoffman-LaRoche for $300,000,000. What is the goal of PCR?.

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Polymerase Chain Reaction

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  1. Polymerase Chain Reaction a.k.a. “How’d they get all that DNA from just a little blood?”

  2. What does PCR stand for? • Polymerase Chain Reaction • Developed by Kary Mullis – Nobel Prize • Received a $20,000 bonus; later sold it to Hoffman-LaRoche for $300,000,000.

  3. What is the goal of PCR? • To make many copies of a small section of DNA. • DNA Synthesis in vitro (in a test tube).

  4. How does PCR work? • Very Similar to DNA Synthesis

  5. How does PCR work? • http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html • http://www.iupui.edu/~wellsctr/MMIA/htm/animations.htm • DNALC • http://wps.prenhall.com/wps/media/objects/487/498929/CDA12_2/CDA12_2a/CDA12_2a.htm

  6. What is needed for a PCR reaction? • DNA template – DNA to be copied; “target sequence”

  7. What is needed for a PCR reaction? • PCR primers – short DNA sequences that bind to DNA; are complimentary to beginning and end of target sequence

  8. What is needed for a PCR reaction? • Taq Polymerase • Polymerase copies DNA • From heat loving bacteria - Thermus aquaticus. • Can survive hot temperatures needed in PCR reaction. • Always copies in 5’  3’ direction

  9. What is needed for a PCR reaction? • Nucleotides – building blocks of DNA • Thermalcycler – computerized to change temperatures

  10. What are the 3 main steps of PCR? • Denaturing – DNA strands separate at hydrogen bonds • Temperature = 950C

  11. What are the 3 main steps of PCR? • Annealing – Primers bind • Temperature is dependent on primer sequence • Annealing temperature = 3(G+C) + 2(A+T) • # of H-bonds between nucleotides

  12. What are the 3 main steps of PCR? • Extension – DNA is copied • Temperature = 720C

  13. PCR Reaction Summary 1. Denaturing 2. Annealing 3. Extension 30 cycles

  14. How many times can target DNA be copied? • DNALC

  15. What would someone do with PCR? • Forensics • Identification of corpses or body parts. • Analysis of pathogens. • Eliminate or link suspects to scene of crime through a sample (blood, human hair, skin, semen) left at scene or circumstantial links (pet hair, plant parts).

  16. What would someone do with PCR? • Medical (genotyping) • Genetic diseases identified pre- or post-natally. • Cancers identified and classified. • Identifying bacterial or viral strains for proper treatment.

  17. What would someone do with PCR? • Food Science • Samples can be tested for genetic engineering traits. • Bacterial contaminants and source of contamination can be quickly identified. • Pedigrees and traits of valuable animal food stocks can be verified.

  18. What would someone do with PCR? • Relationships • Paternity. • Evolution – developing relationships between organisms or studying extinct species from museum specimens. • Ecology – tracking organisms or defining biodiversity by identifying unique species.

  19. PCR Review Music Video BioRad “When You Need to Find Out Who The Daddy Is” BioRad “GTCA”

  20. PCR Lab #1 - Alu • What DNA sequence are we going to copy? • Alu element • Chromosome 16 • Does NOT code for a gene

  21. PCR Lab #1 - Alu • 95% of the human genome does NOT code for proteins • “Junk” DNA • Nobody knows why we have it • All of it is copied during DNA synthesis and passed from one generation to the next

  22. PCR Lab #1 - Alu • Alu is an example of a “jumping gene” (also known as a transposon) • Come from a virus infection • All primates share the same initial Alu sequence • Human chromosomes contain an estimated 1,000,000 Alu copies equaling 10% of the total genome.

  23. PCR Lab #1 - Alu • Alu elements are dimorphic (meaning two forms) • Chromosome 16 either has this Alu element or it doesn’t • Since chromosomes come in pairs, a person is either ++, +-, or -- . • DNA sequences such as Alu are used in diagnosis of genetic disease, forensic identification, and paternity testing.

  24. PCR Lab #1 – PTC Taster • What DNA sequence are we going to copy? • PTC taste receptor gene (produces a bitter taste)

  25. PCR Lab #1 – PTC Taster • Chance discovery

  26. PCR Lab #1 – PTC Taster • Being a “taster” is a dominant trait • Homozygous dominant – TT • Heterozygous – Tt • Homozygous recessive - tt TASTERS NON-TASTERS

  27. PCR Lab #1 – PTC Taster • About 70% of people are tasters • 58% for Aboriginal people of Australia • 98% of Native Americans

  28. PCR Lab #1 – PTC Taster 221 bp • Non-taster PCR product - Taster PCR product - 221 bp SNP – Single Nucleotide Polymorphism. One “letter” makes a big difference.

  29. PCR Lab #1 – PTC Taster • What will be our DNA source? • Cheek cells • Rinse our mouths with a saline solution - (0.8% NaCl)

  30. PCR Lab #1 – PTC Taster • Why not just use pure water to rinse our mouths? • What would happen to our cells in 100% water?) Due to osmosis, cells would burst before we could collect DNA

  31. PCR Lab #1 – PTC Taster • Cells are collected, allowed to settle, and then mixed with Chelex solution. • Negatively charged beads – removes Mg2+ which inhibits Taq polymerase.

  32. PCR Lab #1 – PTC Taster • Chelex, cheek cell mixture is heated to 980C. Heat bursts cells open and cell debris is bound to Chelex beads. • Chelex beads and cell debris is heavy so it will settle to bottom of tube. • We will collect the supernatant (contains DNA) and will freeze it. • Set up PCR reaction on Thursday.

  33. 3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’ 5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’ Denature; 950C Taq Pol 3’ A T C G G A C C G A C T G C A T G C C A G T T A A T A G C C A A T 5’ Anneal; ~600C Extend; 720C 5’ C T G G 3’ 3’ T A G C 5’ Taq Pol 5’ T A G C C T G G C T G A C G T A C G G T C A A T T A T C G G T T A 3’

  34. PCR Lab #2 - mtDNA • What DNA sequence are we going to copy? • mtDNA control element • Does NOT code for a gene

  35. PCR Lab #2 - mtDNA • In which organelle is this DNA stored? • mitochondria • All DNA is NOT stored in nucleus!

  36. PCR Lab #2 - mtDNA • How many genes are on this DNA? • 37 genes in 16,569bp

  37. PCR Lab #2 - mtDNA • What proteins do these genes make? • Proteins involved in cellular respiration

  38. PCR Lab #2 - mtDNA • Interesting facts regarding mtDNA • Always inherited from mom • Mutates at a constant rate

  39. PCR Lab #2 - mtDNA

  40. PCR Lab #2 - mtDNA • Compare sequences to determine genetic relationships (phylogenetic tree)

  41. PCR Lab #2 - mtDNA • Interesting facts regarding mtDNA • Mitochondria may have been a bacterial cell at some point

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