Genetic mutation and analysis Flashcards Preview

PoD > Genetic mutation and analysis > Flashcards

Flashcards in Genetic mutation and analysis Deck (18):

Genetic mutation can be either

germline or somatic


Mutations can be associated with

genes or disease



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



- 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



- 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


Advantages and disadvantages of DNA sequencing

+ Automation and high throughput
- Expensive equipment, poor quality sequence read (deterioration after 700-900 bases)


Problems with next generation sequencing

- Extremely complex task where you need to predict what faulty genes are and aren't going to be involved in, say, cancer
- Can give rise to more confusion and you cannot be sure whether you're predicting a predisposition or an actual cancer


How to choose a test

- Direct test
- Quick and easy
- Cheap
- Sensitivity
- Specificity