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DNA Van Program

DNA Van Program. University of Missouri Columbia . Thanks to Dr. Miriam Golomb AND NATIONAL STARCH COMPANY. TPA-25 ALU DNA Fingerprinting and PCR TM VAN PROGRAM. INSTRUCTOR: SUSIE HELWIG NORTH KANSAS CITY HIGH SCHOOL.

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DNA Van Program

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  1. DNA Van Program University of Missouri Columbia Thanks to Dr. Miriam Golomb AND NATIONAL STARCH COMPANY

  2. TPA-25 ALU DNA Fingerprinting and PCRTMVAN PROGRAM • INSTRUCTOR: • SUSIE HELWIG • NORTH KANSAS CITY HIGH SCHOOL

  3. Why teach Polymerase Chain Reaction (PCR) and DNA Fingerprinting? • Powerful teaching tool • Real-world connections • Link to careers and industry • Laboratory extensions • Hook Math, English and History students into Biology

  4. PCR Procedures Day 1 Day 2 Day 3

  5. Introduction Extract genomic DNA and prepare PCR samples Cycle samples Agarose gel analysis Hardy-Weinberg analysis TPA-25/D17S5

  6. What will we learn with the TPA-25 laboratory? • Introduce the polymerase chain reaction (PCR) technique • Apply PCR to population genetics • Directly measure human diversity at the molecular level

  7. What is PCR? • PCR stands for Polymerase Chain Reaction • DNA replication gone crazy in a tube! • Devised by Kary Mullis in the 1980’s it is a simple and powerful way of making unlimited numbers of copies of a DNA template. • A single DNA molecule can be replicated a billion fold in a few hours. This allows for the resurrection of DNA from fossils like a Neanderthal. • Uses Taq Polymerase heat-resistant DNA polymerase from Thermus aquaticus

  8. What are practical uses for PCR technology in society today? • A single DNA molecule can be replicated a billion fold in a few hours. This allows for the resurrection of DNA from fossils like a Neanderthal. • Small amounts of DNA left at crime scenes can be lifted and analyzed to find the criminal. • Paternity testing and Population genetics.

  9. What is TAQ Polymerase? • Taq is a nickname for Thermus aquaticus, a bacterium that survives and reproduces in hot springs • Taq polymerase is is derived from bacteria that live in hot springs and are among the few enzymes that can function at very high temperatures. Withstands temperatures up to 95°C • Unlike other polymerase Taq can survive in extreme hot temperatures and the enzyme does not denature like most. • It is thermophilic because it loves heat. • This has become a great break through in PCRbecause it can survive the high heat needed.

  10. DNA extraction Cell membrane Nuclear membrane Mg++ Genomic DNA Mg++ Mg++ Heat disrupts membranes Mg++ Mg++ Chelex binds released cellular Mg++ Mg++

  11. Micropipette Use • Twist dial to desired volume. Be careful • this is where you can break these. • Pick up pipette tip by pressing firmly on • the tip located in the box. • Press plunger to first, soft stop • Insert pipette tip into solution to be • transferred • Slowly release plunger to retrieve liquid • Move pipette tip into desired tube • Press plunger past first stop to second, • hard stop to transfer liquid

  12. LOADING DNA IN WELLS BY PIPETTING • MAKE SURE THE PIPETTE READS THE CORRECT AMOUNT TO BE ADDED TO THE WELL. • PRESS THE PLUNGER TO THE FIRST STOP AND SLOWLY RELEASE TO EXTRACT THE DNA FROM THE TUBE. • CAREFULLY ADD DNA BY INSERTING THE TIP OF THE PIPETTE INTO THE BUFFER IN THE MIDDLE OF THE WELL. • BE CAREFUL NOT TO TOUCH THE BOTTOM OF THE WELL. • PRESS THE PLUNGER TO THE FIRST STOP ONLY AND RELEASE THE DNA INTO THE WELL. DO NOT PRESS TO THE SECOND STOP. THIS WILL RELEASE AIR INTO THE WELL. KEEP THE PLUNGER DOWN AT THE FIRST STOP AND TAKE THE PIPETTOR OUT OF THE BUFFER. THIS WILL ENSURE THAT YOU DON’T SUCK THE DNA BACK INTO THE PIPETTE.

  13. Isolation of Cheek Cell DNA • Cheek cells will first be collected by rinsing out your mouth with a saline solution. • Why saline? • The salt keeps the cells in proper osmotic balance so they don’t burst. (Isotonic) • The cells are then spun in a centrifuge and boiled with a resin (CHELEX). Chelex is an ion-exchange resin. • Boiling causes the cells to disrupt and the DNA (now in single-stranded form) extracted in water. • The chelex removes impurities that would interfere with PCR. • The Chelex™ protects the sample from DNAases* that might remain after the boiling and could subsequently contaminate the samples

  14. What does CHELEX do? • The chelex removes impurities that would interfere with PCR. • The Chelex™ protects the sample from DNAases* that might remain after the boiling and could subsequently contaminate the samples. • DNAases are enzymes which occur naturally in all body tissues. • They cut DNA, rendering it unsuitable for PCR. Magnesium ions are essential cofactors for DNAases. • Chelex™ resin binds with cations including Mg+. By binding with the magnesium ions, the Chelex™ resin renders DNAases inoperable, thus protecting DNA from their action.

  15. What does the PCR mix contain? • PCR mix contains • reagents needed for PCR amplification • red and yellow loading dyes • glycerol to make samples sink • Amplified samples can be loaded directly onto agarose gels

  16. What is needed for PCR? • Template (the DNA you are exploring) • Sequence-specific primers flanking the target sequence • Forward • Reverse • Nucleotides (dATP, dCTP, dGTP, dTTP) • Magnesium chloride (enzyme cofactor) • Buffer, containing salt, maintains tonicity, regulation of ions and pH. • Taq Polymerase is responsible for adding nucleotides to the newly formed DNA strand.

  17. How does PCR work? • Heat (94o-C) to denature DNA strands • Cool (64oC) to anneal primers to template • Warm (72oC) to activate Taq Polymerase, which extends primers and replicates DNA • Repeat multiple cycles (30)

  18. 3’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ Denaturing Template Heat causes DNA strands to separate Denature DNA strands 94oC

  19. 5’ 3’ 5’ 3’ 5’ 3’ Annealing Primers • Primers bind to the template sequence • Taq Polymerase recognizes double-stranded substrate 3’ 5’ Primers anneal 58oC 5’ 3’ 5’ 3’

  20. 3’ 3’ 5’ 5’ 5’ 5’ 3’ 3’ Taq Polymerase Extends • Taq Polymerase extends primer • DNA is replicated 5’ 3’ 5’ 3’ TAQ POLYMERASE Extend 72oC 5’ 3’ 5’ 3’ Repeat denaturing, annealing, and extending 30 cycles

  21. 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ The target product is made in the third cycle 3’ 5’ Cycle 1 5’ 3’ Cycle 2 3’ Cycle 3

  22. Amplified Region 5’ Alu 3’ Exon 8 Exon 9 Exon 10 Intron 8 The target sequence • Chromosome 8 • Intron of tissue plasminogen activator (TPA) gene • Alu-TPA25

  23. Alu-TPA25 • The part of your DNA that actually codes for anything is only about 5% of your total chromosomal DNA or genome. • The remaining 95% consists of stretches between genes, and interrupting sequences within genes (introns). Much of this non-coding DNA is thought to be “junk” in that it doesn’t affect phenotype. • This junk “ALU” makes up about 5% genomic DNA as much as all our genes put together. • The presence of ALU sequence in our chromosomes is thanks to an ancient retrovirus which once infected our ancestors. This virus a distant relative of the AIDS virus, copied cellular RNA sequences into DNA and stuck them in at random chromosomal locations.

  24. Alu-TPA25 (continued) • One such ALU sequence is called TPA-25 and is found within an intron of the gene for tissue plasminogen activator. The TPA-25 gene encodes a protein that prevents blood clotting inside tissue.) • Since it is located within a non-coding protein of a gene it doesn’t affect gene expression. • This Alu insertion seems to have happened in the past million years, in a recent human ancestor. As a result some human chromosomes have it and others don’t.

  25. Amplified Region 5’ Exon 8 Alu 3’ Exon 9 Exon 10 Intron 8 PCR Results…. • Alu-TPA25 is dimorphic so there are two possible PCR products: • 100 bp • 400 bp No insertion: 400 bp Alu insertion: 400 bp 330 bp each 300 bp Alu insert

  26. Actual Alu-PCR Results - +/- + 400 bp 100 bp + - +/-

  27. Alu repeats • Occurs >500,000 times in the human haploid genome • Named for the Alu I restriction site within the element

  28. Evolutionary Significance of Alu-TPA 25 Highly conserved Inserted in the last 1,000,000 years Dimorphic (+/+, +/-, -/- ) Used in population genetics, paternity analysis, and forensics

  29. Amplify Alu-region from representative sample population Calculate the expected allelic and genotypic frequencies Perform Chi-squared Test To estimate frequency of Alu within a population:

  30. Alu and Population Genetics Hardy-Weinberg Equilibrium p2 + 2pq + q2 = 1 q p pp pq p +/+ = p2 +/- = 2pq -/- = q2 q pq qq

  31. Calculating Observed Genotypic Frequencies Genotype+/+ (p2)+/- (2pq)-/-(q2)Total (N) # of people9 16 13 38 Observed frequency 0.24 0.42 0.34 1.00 Calculation: +/+ genotypic frequency =# with genotype total number of people (N) =9/38 = 0.24

  32. Calculating Allelic Frequencies • Frequency of p = number of p alleles = 34 = 0.45 • total alleles 76 • Number of p alleles = • +/+ = 9 with two + alleles = 18 + alleles • +/- = 16 with one + alleles = 16 + alleles • Total = 34 + alleles • Total number of alleles = 2N = 2(38) = 76

  33. Using Hardy-Weinberg Determine p2,2pq, and q2 values= Expected genotypic frequencies p = 0.45 , so q = 0.55 since p + q = 1p2 + 2pq + q2 = 1 (0.45)2 + 2 (0.45)(0.55) + (0.55)2 = 1 0.20 + 0.50 + 0.30 = 1 p2 = 0.20 2pq = 0.50 q2 = 0.30

  34. Calculate Expected Number of Genotypes Expected number of genotype = Genotypic frequency x population number (N) GenotypeExpected number +/+ 0.20 x 38 = 8 +/- 0.50 x 38 = 19 -/- 0.30 x 38 = 11

  35. Chi Squared Test ObservedExpected(O-E)2 E +/+980.13 +/-16190.47 -/-13 11 0.36 Total0.96 X2 Critical Value (from statistics table) = 5.9 0.96 falls below 5.9 so the ratio is accepted.

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