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PCR lab Week 1 LE 16-8 Sugar Adenine (A) Sugar Thymine (T) Sugar Sugar Guanine (G) Cytosine (C) DNA structure LE 16-7 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm Key features of DNA structure 5 end Partial chemical structure Space-filling model LE 16-5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate Sugar (deoxyribose) 3 end DNA nucleotide Guanine (G) 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 Hydrogen bonding between DNA bases • A with T, C with G • CG pairs have 3 bonds, AT have two 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. 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. 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. 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 backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand. Cast and Mold- each can copy the other 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 LE 16-10 Parent cell 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. First replication Second replication 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 LE 16-11 Bacteria cultured in medium containing 15N DNA sample centrifuged after 20 min (after first replication) Bacteria transferred to medium containing 14N DNA sample centrifuged after 40 min (after second replication) First replication Conservative model Semiconservative model Dispersive model Less dense More dense Second replication 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 PCR • • • • Polymerase Chain Reaction Uses Taq polymerase Taq= Thermophilus aquaticus PCR amplifies DNA samples 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 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! dNTPs • Individual DNA nucleotides • Four kinds- A, C, G, and T • They match up with template DNA Taq Polymerase • DNA polymerase isolated from Thermophilus aquaticus bacteria • Lives in hot springsheat resistant • Optimal Taq temp- 72C 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 Thermal Cycler • Melting point of DNA= ~94C • Annealing temp = 55C • Optimal Taq polymerase temp= 72C PCR II February 1, 2008 DNA, replication, and PCR 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. 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. 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 PCR • • • • Polymerase Chain Reaction Uses Taq polymerase Taq= Thermophilus aquaticus PCR amplifies DNA samples 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 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 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”) 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 Loading a Gel • DNA is mixed with loading dye • Dye-DNA mixture is placed into gel wells 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 A Jar of Marbles • Space in between the marbles would allow sand to fall • Large grains would fall slower Detection- DNA Staining in Gel • Ethidium bromide is used • Intercalates DNA • Fluorescent under UV light • Intercalates DNA DNA Intercalation • Ethidium bromide sticks between the rungs of the DNA ladder • Can impair proper DNA replication • Wear gloves, please 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 Purifying DNA • Desired DNA fragments can be cut directly from gel, purified, and used 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… The Eukaryotic genome • Human DNA is >99% identical • The PV92 locus of chromosome 16 is dimorphic • Some people have an Alu repeat Eukaryotic Genomes Contain introns Introns are spliced out during translation 5 Exon Pre-mRNA Intron Exon Intron Exon 3 5 Cap Poly-A tail 1 30 31 Coding segment 104 105 146 Introns cut out and exons spliced together Poly-A tail 5 Cap 5 UTR 1 146 3 UTR The Eukaryotic Genome • Contains introns • Introns are spliced out (Retro-)transposons move around the genome across many generations 19.16 Mammals 25-50% Primates Alu 10% 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….) 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? 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!