1. PCR lab Week 1

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

3. DNA structure

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

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

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 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 first step in replication is separation of the two DNA strands.

10. LE 16-9_3 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 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.

11. LE 16-9_4 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 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 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. LE 16-10 Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. 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. Parent cell First replication Second replication

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. LE 16-11 Bacteria cultured in medium containing 15 N DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) Bacteria transferred to medium containing 14 N Less dense More dense Conservative model First replication Semiconservative model Second replication 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 6.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 1 st 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” 4 th Band from top in lane M=500 bp 5 th Band is 400 bp How big are the bands in lane 8?

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

43. 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…

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

45. Eukaryotic Genomes Contain introns Introns are spliced out during translation 5 ExonIntronExonIntronExon 3 Pre-mRNA 13031104105146 Coding segment Introns cut out and exons spliced together 1146 5  Cap Poly-A tail 5 3 UTR

46. The Eukaryotic Genome Contains introns Introns are spliced out

47. (Retro-)transposons move around the genome across many generations 19.16 Mammals 25-50% Primates Alu 10%

48. 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….)

49. 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?

50. 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!