Detecting mutations Lecture 3 Strachan and Read Chapters 16 & 18.

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 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)

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.