DNA Replication/ PCR/ Restriction Digests
Objectives of this Lab Exercise (over the next couple
of weeks):
Polymerase Chain Reaction (PCR)
Restriction Enzyme Digestion
Gel Electrophoresis
Today: Extract the DNA and set up the PCR reactions
DNA Replication
Identify your specific genotype for the “PTC” tasting
gene
The Polymerase Chain Reaction (PCR)
Taq polymerase
Thermus aquaticus
Producing identical copies of
a DNA segment
(typically smaller than a gene)
DNA amplification
Thermocycler
Ingredients
Steps
The Structure of DNA
1) How is the genetic information copied?
2) How does DNA store the genetic
information?
3) How is the genetic information passed
from generation to generation?
The Structure of a Single DNA Strand
o Sugar-phosphate backbone
o Monomers: Nucleotides
o Nitrogenous bases:
purines & pyrimidines
o 5’ and 3’ ends
The Double Helix Base-Pairing
Uniform Width of the DNA Double Helix
The Double Helix
Base-Pairing/
Provided a basis for copying DNA but
the mechanism was not understood
What type of
bonds connects
two nucleotides
together?
Three Alternative Hypothesis
Conservative Replication
Semi-Conservative Replication
Dispersive Replication
Meselson and Stahl’s Experiment
Density Gradient Centrifugation
100% (saturated) NaCl
50% saturated NaCl
25% saturated NaCl
dH2O
Meselson and Stahl’s Experiment
Density Gradient Centrifugation
Red: 15N
Blue: 14N
Manipulation of DNA
Molecular Biology Techniques
Restriction enzymes: endonucleases that recognize a unique nucleotide sequence and make
cuts in the sugar-phosphate backbone of each DNA strand
Single Nucleotide Polymorphisms
(SNPs)
One nucleotide difference in a DNA sequence among individuals
G G C G G G C A C T
G G C G G C C A C T
Can be used for genotyping
DNA Replication
Overview
Origins of Replication - Eukaryotes
May have hundreds or even thousands of replication origins
Replication begins at specific sites where parent strands separate to form replication
bubbles
Replication bubbles expand laterally as DNA replication proceeds
Replication bubbles fuse and synthesis of the daughter strands is complete
sister chromatids!!!
How about
prokaryotes?
The Cast of Characters
Elongation of new DNA at a replication fork:
DNA polymerases add nucleotides to the 3 end of a growing strand
The Role of DNA Polymerase
How does the antiparallel structure of the double helix affect
replication?
DNA polymerases add nucleotides only to the free 3 end of a growing strand
Along one template strand of DNA, the leading strand,
DNA polymerase III can synthesize a complementary strand
continuously, moving toward the replication fork
To elongate the other new strand of DNA, the lagging strand
DNA polymerase III must work in the direction away from the
replication fork
The lagging strand is synthesized as a series of segments called
Okazaki fragments, which are joined together by DNA ligase
Priming Replication
DNA Polymerases cannot initiate replication!
They can only add nucleotides to the 3’end of a growing strand
Need to have a ‘primer’ in place
RNA primer
Only one primer is needed for replication of the leading strand!
For the lagging strand, each Okazaki fragment must have its own primer
SSBPsHelicase
Topoisomerase
The leading strands are faded out
to help you focus on synthesis
of the lagging strand
SSBPs HelicaseTopoisomerase
The leading strands are faded out
to help you focus on synthesis
of the lagging strand
1. Primer added. Primase synthesizes RNA primer.
Primase
RNA
primer
2. First fragment synthesized. DNA polymerase III works in 5 → 3 direction,synthesizing first Okazaki fragment of lagging strand.
DNA polymerase III
Sliding clamp
Okazaki fragment
3. Second fragment synthesized. Primase and DNA polymerase IIIsynthesize another Okazaki fragment.
1st Okazaki fragment
2nd Okazaki fragment
4. Primer replaced. DNA polymerase I removes ribonucleotides of primer,replaces them with deoxyribonucleotides in 5 → 3 direction.
DNA polymerase I
5. Gap closed. DNA ligase closes gap in sugar-phosphate backbone.
DNA ligase
Telomeres Shorten during Normal DNA Replication
1. Helicase unwinds end of DNA helix (at end of chromosome).
5
5
3
3
CHROMOSOME SHORTENING DURING NORMAL DNA REPLICATION
RNA primer
33 5
SSBPs Helicase
Leading strand
Lagging strand
Sliding clampDNA polymerase
5
5
End of chromosome
2. DNA polymerase completes the leading strand. Primase synthesizes RNA primer at endof lagging strand.
Telomerase Prevents Shortening of Telomeres
5
DNA polymerase
TELOMERE REPLICATION
Missing DNA on
lagging strand
Sliding clamp
55
RNA primer
3
3
53
3Telomerase with itsown RNA template
5
5
5
3
3
1. When the RNA primer is removed from the5 end of the lagging strand (see Figure 14.14),a strand of parent DNA remains unreplicated.
4. Primase, DNA polymerase, and ligase thensynthesize the lagging strand in the 5→3direction, restoring the original length of thechromosome.
2. Telomerase binds to the “overhanging” sectionof single-stranded DNA. Telomerase addsdeoxyribonucleotides to the end of the parentDNA, extending it.
3. Telomerase moves down the DNA strand andadds additional repeats.
The Cast of Characters
ampR
• aproximately 5,400 bp
Bacterial Transformation
GFP are used to monitor the activity of altered genes
Transformation
experiment
Gene CloningCLONING -- Producing identical copies of a DNA segment
(gene)
Figure 20.2