Genetic mutation and analysis Flashcards
Genetic mutation can be either
germline or somatic
Mutations can be associated with
genes or disease
Polymorphism
Difference in genetic code from person to person but usually there is no phenotypic effect
Types of mutation
- Coding
- Non-coding
Coding mutations
- Silent. CGA to CGC is still arginine
- Missense. CGA (Arg) to GGA (Gly)
- Nonsense. CGA (Arg) to TGA (Stop)
- Frameshift (deletion/insertion). CGA (Arg) to CCGA (Pro, then out of frame). Likely to hit a stop codon by chance
Nomenclature of protein sequence mutation
- M is mutation
- X is stop codon
- Del is deletion
Nomenclature of DNA sequence
- Intronic nucleotides near the start of the intron have number of last nucleotide of the preceding exon, a plus sign and position in the intron c.77_1G
- Near end: number of the first nucleotide of the following exon, a minus sign and the position upstream in the intron e.g. c.78-1G
Methods of detecting mutation
- Polymerase chain reaction
- Amplification refractory mutation system (ARMS)
- Gel electrophoresis
- Restriction fragment length polymorphism (RFLP) analysis
- DNA sequencing
PCR
- In vitro
- Denature, heat -> anneal -> extend, heat resistant polymerase used -> repeat tens of times
- Advantage: quick, ease of use, sensitive, robust
- Applications: DNA cloning, sequencing, gene identification, gene expression studies, forensic medicine, detection of mutations
ARMS
- Amplification refractory mutation system
- Using PCR but with different primers
- If you use a normal primer and you see an amplification then there’s a wild-type allele
- If you use a mutant primer and there’s amplification, there’s a mutant allele
Advantages and disadvantages of ARMS
+ Cheap, labelling not required
- Electrophoresis required, primer design critical, need to know nature of mutation you’re looking for, need to sequence info, limited amplification size, infidelity of DNA replication could give rise to a mutation
Gel electrophoresis
- Separate DNA fragments by size
- Apply electric field
- DNA is negatively charged
- Separate through agarose gel matrix
- Visualise DNA fragments
RFLP analysis
- Restriction fragment length polymorphism
- DNA is broken into pieces and digested by restriction enzymes, the resulting restriction fragments are separated according to their lengths by gel electrophoresis
Advantages and disadvantages of RFLP
+ Simple, cheap, non-radioactive
- Requires gel electrophorsesis, not always feasible, need to know exact characterisation of mutation
DNA sequencing
- Gold standard for detection
- You can look for errors in entire DNA fragment
- Chain termination methods: uses dideoxynucleotides
