Basic methods in genetics

PCR; Polymerase Chain Reaction

Restriction enzyme digestions
Gel electrophoresis

PCR; Polymerase Chain Reaction

Amplification of specific DNA sequences

Invented by Kary Mullis in 1983
Revolutionized the world of molecular biology
Mimics cells own DNA replication machinery

 Ingredients in PCR:
DNA as a template
Thermo stable DNA polymerase enzyme
Deoxynucleoside triphosphates (dNTPs)
Synthetic oligonucleotide primers
The flanking sequence of the target locus
needs to be known!

 Three major steps in PCR:

Denaturation: Strand separation at 95C
Annealing: Hybridization of primers at 45-60C
Extension: DNA synthesis at 72C
Three steps are repeated for 25 to 40 times
Final elongation step at 72C
Exponential increase of the number of copies millions
of copies of the target sequence

Primers

DNA-Polymerase
+ Nucleotides

Steps in PCR
Denaturation 95C
Annealing 50-60C

Extension 72C

Denaturation,
annealing
x30
Extension

Technical problems

Contamination
Sensitivity to the levels of divalent cations
Quality of template DNA
Limited size of amplified product:
300 bp-1000 bp are most efficient
possible to amplify fragments of several kb

Primer design

5GATACCAGATGCATACGGCAACTAT 3

Must be very specific

3TATCAACGGCATACGTAGACCATAG 5
No primer-primer interactions
No hairpin formation
GATGATGCATCTAG 5
A
CTACTAGAGG 3
No self-annealing

DNA sequence from the database (or from sequencing)

ENSEMBL
BLAST

http://www.ensembl.org/
http://www.ncbi.nlm.nih.gov/BLAST/

Primer3 program:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

What are enzymes?

Proteins that speed up chemical reactions in the body=
protein catalyst
essential to sustain life
Substrate = The molecule with which an enzyme
interacts
Enzyme function is highly dependent on environmental
characteristics such as temperature and pH.

Restriction enzymes

Nucleases:
exonucleases: remove nucleotides from the end of DNA or
RNA
endonucleases: make cuts at internal phosphodiester
bonds
Restriction endonucleases:
Discovered in the late 1960s
Found and purified from bacteria
Three types:
Type I and III: do not recognize a specific sequence to cut
Type II: cut specific recognized sequence

Type II enzymes:
Over 2500 different enzymes have been isolated
More than 500 enzymes are commercially available
Cut often sequence at palindromic hexanucleotide
sequences
e.g. EcoRI : GAATTC

CTTAAG
Most enzymes cut within the recognition sequence
Leaves sticky or blunt ends

A sticky end:

Restriction
enzyme
cuts here

G
C
C
A
A
T
T
C
G
A

C
G
G
T
T
A
A
G
C
T

Restriction
enzyme
cuts here

Strands
separate

G C
C G
C G

C G
G C
A T

Each strand
has a sticky
end

A blunt end:

Restriction
enzyme
cuts here

G
C
C
A
A
T
T
C
G
A

C
G
G
T
T
A
A
G
C
T

Restriction
enzyme
cuts here

Strands
separate

G
C
C
A
A

C
G
G
T
T

T
T
C
G
A

Each strand
has a blunt
end

A
A
G
C
T

Restriction enzymes are powerful tools for

molecular genetics
Restriction enzymes can be used to:
Create restriction maps
Analyze sequence variations
Rearrange DNA molecules
Create mutants
Analyze the modification status of the DNA
...other applications

The designing of digestion reactions:

Sequence of the target DNA must be known
Webcutter 2.0:
http://www.firstmarket.com/cutter/cut2.html

Gel electrophoresis
A method that separates macromolecules on the basis of:
size
electric charge
other physical properties

Gel acts as a support medium

Electric field is generated across the gel
DNA is negatively charged migration towards the
positive pole
Small molecules move faster than big molecules
Ethidium bromide staining
Intercalates between bases of DNA
Can be visualized under UV-light

Requirements in gel electrophoresis:

An electrophoresis chamber and power supply

Gel casting tray
Sample comb
Agarose
Electrophoresis buffer
Loading buffer
DNA ladder (= size standard)
Ethidium bromide

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