Detecting mutations

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Detecting mutations

• Lecture 3
• Strachan and Read Chapters 16 18

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Proving it's the right gene

• Genetic evidence is the "gold standard" for
  deciding if your candidate gene is the correct
  one. The questions to be answered are
• Is there a mutation in the gene, that affects
  protein structure or gene expression?
• Is the mutation found in patients but not healthy
  controls?
• Do some patients have a different mutation in the
  same gene?
• In the case of complex disease, this is hard to
  prove - because the same disease may have
  different genetic causes (heterogeneity)

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Methods for mutation detection

• Deletions, insertions, or re-arrangements (gt10bp)
  can be detected by restriction enzyme digestion,
  gel electrophoresis, Southern blotting and
  probing with the candidate gene, or by PCR of
  regions of the candidate gene
• This was used to find the mutations causing
  myotonic dystrophy and Huntingtons Disease

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Myotonic dystrophy

• Autosomal dominant neuromuscular disease
• Main symptoms muscle weakness, wasting, myotonia
  (cant relax grip)
• Can be fatal in infants
• Affects up to 1/8000 people (commonest adult
  muscular dystrophy, similar number at risk
• Affects also eyes, endocrine organs, heart, brain
• Anticipation earlier onset, more severe, in
  successive generations
• In 1982, mapped to chromosome 19 gene discovered
  in 1992

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Huntingtons disease

• Autosomal dominant, affects 1/20000 plus more at
  risk
• Progressive brain degeneration, due to death of
  certain groups of neurons
• Onset usually late 30s, death 15 years later
• Symptoms personality changes, memory loss,
  movement disorder (jerkiness), chronic weight
  loss
• No treatment or cure
• In 1983, mapped to chromosome 4 gene discovered
  in 1993

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Detecting small mutations

• Small changes such as single base changes or
  insertions/deletions of lt 10bp are harder to
  detect. Small changes such as single base
  mutations can be detected in many ways
• Purify DNA fragment to be analysed, usually by
  PCR. A label (radioactive or fluorescent) can be
  incorporated at this stage.
• You can also start with mRNA, by first
  reverse-transcribing it into cDNA. This saves you
  having to analyse all the non-coding parts of the
  gene (the introns) which are present in genomic
  DNA.
• Treat DNA fragment in some way, which is specific
  to the method being used
• Analyse the products by gel electrophoresis or
  equivalent technique

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SSCP

• In Single-strand conformation polymorphism (SSCP)
  the DNA fragment is heated to denature the
  strands, then cooled rapidly on ice
• Some of the single DNA strands will form
  secondary structures by themselves rather than
  re-annealing with their complementary strand
• The type of secondary structure formed is
  determined by the base sequence, and influences
  the mobility of the fragment on non-denaturing
  acrylamide gel electrophoresis
• A slight difference in mobility relative to a
  normal control fragment indicates a mutation
• Quick and easy to do on a small scale

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Heteroduplex analysis

• If a fragment is PCR-amplified from a sample of
  DNA that is heterozygous for a mutation, the
  product will contain fragments that are different
  at a single position in the sequence
• If they are denatured and renatured, they will
  form either perfectly-matched double stranded
  DNA, or "heteroduplex" DNA in which one strand is
  from the normal and the other from the mutant
• Heteroduplexes have slower mobility on agarose
  gel electrophoresis than perfectly-matched
  sequences
• If the sample to be tested is potentially
  homozygous for the mutation (e.g. in a recessive
  disease) it can be mixed with wild-type DNA
  before PCR
• A new method, Denaturing High-Performance Liquid
  Chromatography (DHPLC), uses the same principle
  but separates the fragments on HPLC columns (very
  quick and accurate)

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Heteroduplex and dHPLC
http//www.uni-saarland.de/fak8/huber/dhplc.htm
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Direct DNA sequencing

• This is the slowest method, but also the most
  definitive
• The fragment is sequenced by the dideoxy method
• A base change is revealed as a position in the
  sequence ladder where there are two bases
  side-by-side instead of the usual one
• This is because the DNA template used for
  sequencing contained a mixture of normal and
  mutant sequences

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1,2 SSCP. 1 is a normal sample, 2 is a mutant.
3,4,5 Heteroduplex analysis. 3, homozygous
normal 4, homozygous for a mutation 5,
heteroduplex formed by mixing normal and mutant.
GATC direct DNA sequencing. Arrow shows
position of mutant base normal allele has A,
mutant has C.