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Flashcards in Genetic mutation and analysis Deck (18):
1

Genetic mutation can be either

germline or somatic

2

Mutations can be associated with

genes or disease

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Polymorphism

Difference in genetic code from person to person but usually there is no phenotypic effect

4

Types of mutation

- Coding
- Non-coding

5

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

6

Nomenclature of protein sequence mutation

- M is mutation
- X is stop codon
- Del is deletion

7

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

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Methods of detecting mutation

- Polymerase chain reaction
- Amplification refractory mutation system (ARMS)
- Gel electrophoresis
- Restriction fragment length polymorphism (RFLP) analysis
- DNA sequencing

9

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

10

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

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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

12

Gel electrophoresis

- Separate DNA fragments by size
- Apply electric field
- DNA is negatively charged
- Separate through agarose gel matrix
- Visualise DNA fragments

13

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

14

Advantages and disadvantages of RFLP

+ Simple, cheap, non-radioactive
- Requires gel electrophorsesis, not always feasible, need to know exact characterisation of mutation

15

DNA sequencing

- Gold standard for detection
- You can look for errors in entire DNA fragment
- Chain termination methods: uses dideoxynucleotides

16

Advantages and disadvantages of DNA sequencing

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

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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

18

How to choose a test

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