Molecular techniques

• Procedure of PCR

  1. Denaturation
  2. primer annealing
  3. extension
• denaturation in pcr

  • heat treatment up to 95 degrees celsius to separate 2 strands of dna (break hydrogen bonds)
• Primer annealing in pcr

  • cooling to 60 degrees celsius
  • presence of large excess of dna primers
  • specific annealing of complementary sequences to 3’ ends of each template dna strand
• extension in pcr

  • taq polymerase synthesises complementary dna strand by catalysing formation of phosphodiester bonds between nucleotides at 72 degrees Celsius
  • dna primer provides 3’ OH group
  • forms single stranded fragment
• advantages of pcr

  • can amplify dna from very small amounts
  • high speed, many copies obtained within a few hours
  • high specificity, amplifies only target sequences (primers are specific)
• Limitations of pcr

  • taq polymerase lacks proofreading ability, errors occurring early will be compounded with every round
  • requires precise knowledge of sequence of target region in order to design primers
  • limited length of 3kb of fragment that can be amplified, increase in length decreases efficiency of amplification, polymerase falls off template
• Gel electrophoresis procedure

  1. dna sample loaded into wells with loading dye
  2. current switched on
  3. after fixed time, gel slab removed, soaked in dna staining dye (ethidium bromide)
  4. bands become visible, illuminated under UV light
• mechanism of gel electrophoresis

  • agarose gel is a mesh work of polymer fibres that impedes movement of larger fragments more than shorter fragments, shorter fragments can move faster towards the anode
  • loading buffer is dense, helps dna sink to bottom of well
  • loading buffer contains dye that travels along the gel with dna, visualise and monitor progress of electrophoresis
  • buffer solution conducts electricity and generates electric field
• usage of dna ladder

  • contains prepared mixtures of dna fragments with known lengths
  • can compare positions of samples with ladder to estimate fragment length and relative amount of dna
• Purpose of southern blotting

  • detect specific nucleotide sequences after gel electrophoresis
• Procedure of Southern blotting

  1. Allow DNA sample to be cut by restriction enzymes

  2. Fragments of sample separated via gel electrophoresis

  3. Gel slab with fragments placed between nitrocellulose membrane and paper towels (top) and absorbent sponge in alkaline solution (bottom)

  4. Paper towels draw alkaline solution up through the gel, denatures and separates double-stranded DNA fragments, single-stranded DNA fragments drawn upwards onto nitrocellulose membrane (binds in same position as gel)

  5. Membrane is incubated with radioactive single-stranded DNA probe (probe consists of single-stranded DNA synthesised to be complementary to part of target sequence, only DNA fragments with target sequence will hybridise to probe)

  6. Membrane washed to remove unhybridised probes

  7. Autoradiography (x-ray film over membrane), radioactivity of bound probes exposes film to form image corresponding to bands base-paired to probe
• Restriction fragment length polymorphism

  • presence of dna polymorphisms in homologous regions of different individuals
• DNA polymorphisms: non-coding sequences at a particular locus that exhibits small nucleotide sequence differences in different individuals
• dna polymorphisms can exist as:

  • differences of a single base-pair (single nucleotide polymorphisms SNPs)
  • varying number of tandem repeats
• RFLP in disease detection:

  • sickle cell anaemia
  • restriction site for MstII eliminated in disease allele
• Procedures for RFLP

  Method 1:

  1. digest dna with restriction enzyme (MstII for sickle cell anaemia specifically) produce fragments of diff lengths
  2. separate by gel electrophoresis
  3. southern blotting, incubate with specific radioactive probes
  4. visualise bands via autography

  Method 2:

  1. design dna primers specific to flanking sequences of the specific gene
  2. amplify with pcr
  3. digest with restriction enzyme (only for disease detection)
  4. separate via gel electrophoresis and stain with ethidium bromide, visualise under uv light