Transcript Finding genes and detecting mutations

Detecting mutations
Lecture 3
Strachan and Read Chapters 16 & 18
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)
Methods for mutation detection
• Deletions, insertions, or re-arrangements
(>10bp) 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
Huntington’s Disease
Myotonic dystrophy
• Autosomal dominant neuromuscular disease
• Main symptoms: muscle weakness, wasting,
myotonia (can’t 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
Huntington’s 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
Detecting small mutations
• Small changes such as single base changes or
insertions/deletions of < 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
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
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 perfectlymatched 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)
Heteroduplex and dHPLC
http://www.uni-saarland.de/fak8/huber/dhplc.htm
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
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.