Basic methods in 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 cells 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 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

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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_w
  ww.cgi

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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 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

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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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A sticky end
Restriction enzyme cuts here
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A blunt end
Restriction enzyme cuts here
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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/c
  ut2.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