Base substitution can be:
Transition and Transversion
Pyrimidine to Pyrimidine
Purine to Purine
Base substitution ‘Transition’
Pyrimidine to Purine
Base substitution ‘Transversion’
Types of mutation?
Silent/synonymous
Missense (conservative & nonconservative)
Nonsense
Framshift
Detection for known mutations:
- Allele specific oligonucleotide hybridization
- Multiplex Ligation Dependent Probe Amplification
- DNA Sequence
- DNA Microarray
- Known mutation
- Also known as the “Dot-Blot” method
- Specific binding of the chromogenic probe to target DNA (with color = mutation)
Allele specific oligonucleotide hybridization
- Known Mutation
- 50 different probes for 50 different genes
- Detects Deletion and Duplication
- unequal peaks = mutation, not amplified
Multiplex Ligation Dependent Probe Amplification
- Known Mutation
- Base on the DNA sequence (compared to normal DNA)
- Different sequence = mutation
- Maxam Gilbert (bottom to top)
- Dye terminator
DNA sequencing
- Known Mutation
- Probes are present in array, samples added to each wells
- Many colors, different samples, different fluorescent
DNA Microarray
- Unknown mutation
- Detects 80-90%
- Fragment size limit 150-200 bp
- Different folding patterns = Different migration pattern = mutation
- secondary structure of sSDNA
Single Strand Conformation Polymorphism
- Unknown Mutation
- modification of AGE
- Uses denaturing chemicals ( Formamide and Urea)
- Top = lowest denaturing condition
- Bottom = highest denaturing condition
Denaturing Gel Gradient Electrophoresis
- Unknown Mutation
- Modification of DGGE
- Use temperature instead of denaturing chemical
- More reproducible and reliable
Temperature Gradient Gel Electrophoresis
- Unknown Mutation
- Uses diff restriction cut sites to detect mutation
- Same principle with DNA Fingerprinting
- Change cut sites (different fragments) = mutation
- Detected mutations affect palindromic cut sites
Restriction Fragment Length Polymorphism
- Unknown Mutation
- Homoduplex and Heteroduplex (bulge)
- No Heteroduplex= no mutation (no bulge)
- Base substitution not easily detected
- Homoduplex stays at the lower portion = mutant
Heteroduplex analysis
- If wild type and homoduplex have the same migration
- Heteroduplex bulge cutted (bands)
- Lane with bands = mutation
Single stranded Specific Nuclease
Detection for unknown mutation:
Single strand conformation polymorphism
Denaturing gel gradient Electrophoresis
Temperature gradient gel Electrophoresis
Restriction Fragment Length Polymorphism
Heteroduplex analysis
