PCR lab

Week 1

LE 16-8

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Sugar

Sugar

Sugar

Sugar

DNA structure

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

LE 16-5Sugar–phosphate

backbone

5 end

Nitrogenousbases

Thymine (T)

Adenine (A)

Cytosine (C)

DNA nucleotidePhosphate

3 endGuanine (G)

Sugar (deoxyribose)

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 back-bones 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

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 moleculeseparate, 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 cellFirstreplication

Secondreplication

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 16-11Bacteriacultured in mediumcontaining15N

DNA samplecentrifugedafter 20 min(after firstreplication)

DNA samplecentrifugedafter 40 min(after secondreplication)

Bacteriatransferred tomediumcontaining14N

Lessdense

Moredense

Conservativemodel

First replication

Semiconservativemodel

Second replication

Dispersivemodel

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. dNTPs2. Mg++ containing Buffer3. Taq polymerase4. Primers for your gene

of interest5. Thermal cycler6. 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 springs- heat 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 in lane

8?

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 intronsIntrons are spliced out during translation

5 Exon Intron Exon Intron Exon 3Pre-mRNA

1 30 31 104 105 146

Codingsegment

Introns cut out andexons spliced together

1 1465Cap

5Cap

Poly-A tail

Poly-A tail

5 3UTR UTR

The Eukaryotic Genome

• Contains introns• Introns are spliced out

(Retro-)transposons move around the genome across many generations

19.16

Mammals25-50%

PrimatesAlu10%

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!