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

2. 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)

3. 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

4. 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

6. 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

8. 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

9. 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

10. 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)

12. Heteroduplex and dHPLC

13. 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

14. 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.