basic lab methods in molecular genetics
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Basic methods in genetics
• PCR; Polymerase Chain Reaction• Restriction enzyme digestions• Gel electrophoresis
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PCR; Polymerase Chain Reaction
• Amplification of specific DNA sequences• Invented by Kary Mullis in 1983• Revolutionized the world of molecular biology• Mimics cell’s own DNA replication machinery
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• 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!
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• Three major steps in PCR:• Denaturation: Strand separation at 95°C
• Annealing: Hybridization of primers at 45-60°C
• Extension: DNA synthesis at 72°C
• Three steps are repeated for 25 to 40 times
• Final elongation step at 72°C
• Exponential increase of the number of copies → millions of copies of the target sequence
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DNA-Polymerase + Nucleotides
Primers
Denaturation 95°C
Annealing 50-60°C
Extension 72°C
Denaturation, annealing
Extension
x30
Steps in PCR
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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
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Primer design
• Must be very specific
• No primer-primer interactions
• No “hairpin” formation
• No self-annealing
• DNA sequence from the database (or from sequencing)• ENSEMBL http://www.ensembl.org/
• BLAST http://www.ncbi.nlm.nih.gov/BLAST/
• Primer3 program:
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
CTACTAGAGG 3´
GATGATGCATCTAG 5´A
3´TATCAACGGCATACGTAGACCATAG 5´
5´GATACCAGATGCATACGGCAACTAT 3´
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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.
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Restriction enzymesNucleases:
• 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
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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
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T A
TA
TA
GC
G C
G C
GC
TA
T AGC
Restrictionenzymecuts here
AA
AA T
T
TA
G C
G CGC
GCGC
TT
Each strand has a ”sticky”end
Strandsseparate
A sticky end:
Restrictionenzymecuts here
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T A
TA
TA
GC
G C
G C
GC
TA
T AGC
Restrictionenzymecuts here
Strandsseparate
TAGC
G C
GC
TA
T A
TAG C
T A
GC
Each strand has a ”blunt”endRestriction
enzymecuts here
A blunt end:
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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
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The designing of digestion reactions:
• Sequence of the target DNA must be known• Webcutter 2.0:
http://www.firstmarket.com/cutter/cut2.html
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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
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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
• Transilluminator