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PCR lab

PCR lab. Week 1. Sugar. LE 16-8. Sugar. Adenine (A). Thymine (T). Sugar. Sugar. Guanine (G). Cytosine (C). DNA structure. 5  end. Hydrogen bond. 3  end. 1 nm. LE 16-7. 3.4 nm. 3  end. 0.34 nm. 5  end. Key features of DNA structure. Partial chemical structure.

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PCR lab

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  1. PCR lab Week 1

  2. Sugar LE 16-8 Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C)

  3. DNA structure

  4. 5 end Hydrogen bond 3 end 1 nm LE 16-7 3.4 nm 3 end 0.34 nm 5 end Key features of DNA structure Partial chemical structure Space-filling model

  5. Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) LE 16-5 Adenine (A) Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose) 3 end Guanine (G)

  6. DNA structure is antiparallel • There is a 3’ end and a 5’ end • Each strand is unidirectional • Many enzymes that replicate DNA are unidirectional also

  7. Hydrogen bonding between DNA bases • A with T, C with G • CG pairs have 3 bonds, AT have two

  8. LE 16-9_1 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

  9. LE 16-9_2 The first step in replication is separation of the two DNA strands. The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

  10. LE 16-9_3 The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

  11. LE 16-9_4 The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

  12. Cast and Mold- each can copy the other

  13. How is DNA replicated? • It was expected, but not proven, that DNA was replicated semiconservatively • Competing models were the conservative model and the dispersive model

  14. Second replication First replication Parent cell Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. LE 16-10 Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple-mentary strand. Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

  15. Meselson-Stahl experiment • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen • The first replication produced a band of hybrid DNA, eliminating the conservative model • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model

  16. Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N Less dense DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) LE 16-11 More dense Second replication First replication Conservative model Semiconservative model Dispersive model

  17. DNA Polymerase • Copies DNA • Requires primers (primase) • Requires unwound DNA (helicase) • These are DNA binding proteins • Works in a unidirectional manner (5’-3’) • PCR uses Taq polymerase

  18. PCR • Polymerase Chain Reaction • Uses Taq polymerase • Taq= Thermophilus aquaticus • PCR amplifies DNA samples

  19. PCR • Step 1- Melting • DNA denatures • Step 2- Annealing • Primers bind to complementary sequences • Step 3- Elongation • Taq DNA polymerase adds free nucleotides to strands • Cycle is complete, DNA has doubled • Process can begin again

  20. Ingredients for PCR 1. dNTPs 2. Mg++ containing Buffer 3. Taq polymerase 4. Primers for your gene of interest 5. Thermal cycler • A gene (piece of DNA) you are interested in All together = DNA xerox machine!

  21. dNTPs • Individual DNA nucleotides • Four kinds- A, C, G, and T • They match up with template DNA

  22. Taq Polymerase • DNA polymerase isolated from Thermophilus aquaticus bacteria • Lives in hot springs- heat resistant • Optimal Taq temp- 72C

  23. Primers • Single-stranded DNA sequences of 15-30 bp specific to gene of interest • One at the 5’ start, the other at the 3’ end of your gene

  24. Thermal Cycler • Melting point of DNA= ~94C • Annealing temp = 55C • Optimal Taq polymerase temp= 72C

  25. PCR II February 1, 2008

  26. DNA, replication, and PCR

  27. DNA Lecture review • DNA subunits are called _______. • They are comprised of a sugar, a ____, and a _______. • There are 4 kinds of DNA bases: __, __, __ and _______. • Adenine always binds with ______ and guanine with_______- this is “______’s rules”. • DNA bases cling together by _____ bonds.

  28. More DNA facts • DNA is the universal code to make ________. • The sides of the DNA ladder run 5’-3’ down one side, and 5’-3’ up the other. This is called _____ structure. • DNA is copied with the enzyme __________. • DNA’s melting point is___________. • People have about ______DNA base pairs per haploid cell.

  29. DNA Polymerase • Copies DNA • Requires primers (primase) • Requires unwound DNA (helicase) • These are DNA binding proteins • Works in a unidirectional manner (5’-3’) • PCR uses Taq polymerase

  30. PCR • Polymerase Chain Reaction • Uses Taq polymerase • Taq= Thermophilus aquaticus • PCR amplifies DNA samples

  31. PCR • Step 1- Melting • DNA denatures • Step 2- Annealing • Primers bind to complementary sequences • Step 3- Elongation • Taq DNA polymerase adds free nucleotides to strands • Cycle is complete, DNA has doubled • Process can begin again

  32. Gel Electrophoresis • Phoresis- “carrying” (G) • Moves (carries) DNA through a gel using electricity • Speed depends on DNA length • Used for isolation, purification, and measurement of DNA fragments

  33. DNA is Negatively Charged • Phosphates each carry a single negative charge • m/Z ratio for all DNA segments is ~equal • DNA will move to (+) electrode (“Run to the red”)

  34. Agarose Gel Purified from seaweed • Porous at molecular level • DNA moves through pores • Buffer conducts electricity • Large DNA molecules move slower than small ones • Density can be varied

  35. Loading a Gel • DNA is mixed with loading dye • Dye-DNA mixture is placed into gel wells

  36. Loading a gel • Put pipette tip in well below buffer level • Depress plunger to 1st stop- avoid bubbles • Remove pipette tip BEFORE releasing plunger • Change tips before loading next well

  37. A Jar of Marbles • Space in between the marbles would allow sand to fall • Large grains would fall slower

  38. Detection- DNA Staining in Gel • Ethidium bromide is used • Intercalates DNA • Fluorescent under UV light • Intercalates DNA

  39. DNA Intercalation • Ethidium bromide sticks between the rungs of the DNA ladder • Can impair proper DNA replication • Wear gloves, please

  40. Sorting DNA by size • Which lane(s) have the largest DNA fragments? The smallest? • What do you think is in lane M? • M is a marker • Also called a “ladder” • 4th Band from top in lane M=500 bp • 5th Band is 400 bp • How big are the bands in lane 8?

  41. Purifying DNA • Desired DNA fragments can be cut directly from gel, purified, and used

  42. What will we find in our DNA? • In order to tell students apart, we must have DNA of different length • We are looking for the “Alu repeat” at one place in the genome (the PV92 locus of chromosome 16) • Some folks got it, some folks don’t • Some folks got it half the time…

  43. The Eukaryotic genome • Human DNA is >99% identical • The PV92 locus of chromosome 16 is dimorphic • Some people have an Alu repeat

  44. Eukaryotic Genomes Contain intronsIntrons are spliced out during translation 5¢ Exon Intron Exon Intron Exon 3¢ Pre-mRNA 5¢Cap Poly-A tail 1 30 31 104 105 146 Introns cut out and exons spliced together Coding segment Poly-A tail 5¢Cap 1 146 UTR 5¢ 3¢ UTR

  45. The Eukaryotic Genome • Contains introns • Introns are spliced out

  46. (Retro-)transposons move around the genome across many generations 19.16 Mammals25-50% PrimatesAlu10%

  47. Much of the Eukaryotic genome is “Junk DNA” • 500,000 Alu sites in the human genome • PV92 on chromosome 16 is just one place were the Alu sequence can be found (sometimes….)

  48. Gene frequencies • If we know how common a gene is, we can predict its distribution in the population • If a coin is flipped twice, what are the odds of getting • 2 heads? • 2 tails? • One of each?

  49. Hardy-Weinberg Equilibrium • Coin flip is based on a “gene frequency” of 50% • Genes do not always have 50% frequency • What if the frequency is 40%? • We can use algebra… • The Hardy-weinberg equation!

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