Lecture 10: Detection of Genetic Variation

Question 1

What is genotyping/mutation detection?

Answer 1

Determination and/or identification of a particular genetic variation

Question 2

Do most genetic variations change the physical properties of their products?

Answer 2

No, they can affect expression levels, when are where it’s expressed and the splice site locations

Question 3

What is the workflow of Genetic tests?

Answer 3

1. Decision to determine the genotype/mutation
2. Collection of DNA via blood/biopsy
3. Appropriate tests conducted
4. Results interpreted

Question 4

What are the 4 critical techniques of genotyping?

Answer 4

• Amplification by PCR
• Restriction Enzyme Digestion
• Separation of fragments by electrophoresis, chromatography or mass spectrometry
• Detection by general detectors (Ethidium Bromide) or specific detectors (radioactive/fluorescent probes)

Question 5

What are the pros and cons of DNA sequencing?

Answer 5

Pros:
- Direct, absolute Identification
- Essential to identify mutations
Cons:
- Expensive
- Time consuming
- Requires skilled operators

Question 6

What is AFLP analysis?

Answer 6

Amplified Fragment Length Polymorphism analysis involves identification of indels >15bps by size

Question 7

What are the pros and cons of AFLP analysis?

Answer 7

Pros:
- Simple to perform
- Cheaps
- Easy to interpret
Cons: 
- Limited to indels bigger than >15bps

Question 8

What is RFLP analysis?

Answer 8

Restriction Fragment Length Polymorphism analysis uses restriction enzymes and separated the fragments

Question 9

Rsa I only cuts DNA at _________.

Answer 9

GTAC

Question 10

What are the pros and cons of RFLP?

Answer 10

Pros:
- Uses internal controls
- Quick
- Easy to interpret
- Cheap
Cons:
- Limited to SNP/mutations within RE sites

Question 11

What are fluorescent probes and primers?

Answer 11

Single-stranded DNA oligonucleotides about 20-40 nucleotides long that often contain a quencher that stops the fluorescent dye from fluorescing

Question 12

What is a FRET assay?

Answer 12

Fluorescent detection with only one colour

Question 13

How does FRET work?

Answer 13

• 2 primers bind to both the wildtype and mutant on either side of a SNP site (when they are close if one is excited by light then so is the other = fluorescence).
• Sample is heated and fluorescence will disappear as they are denatured, the weaker bound (mutant) will go first

Question 14

What is a Taqman assay?

Answer 14

Fluorescent detection with 2 colours

Question 15

How does a Taqman assay occur?

Answer 15

• 2 probes with different dyes at the 5’ end and a quencher at the 3’ end bind to DNA
• during PCR, polymerase 5’ nuclease activity cleaves the dye = fluorescence

Question 16

What are the pros and cons of fluorescent detections?

Answer 16

Pros:
- Closed tube assay
- Quick and easy, can be automated
- Decreased chance for error
Cons:
- Requires expertise in molecular biology
- Probe design is difficult
- Expensive 
- Expensive to set up the lab

Question 17

What is MALDI-TOF MS?

Answer 17

Method of sequencing using Mass Spectrometry that uses ddNTP and known molecular weights of the bases to determine the sequence

Question 18

What is SSCP analysis?

Answer 18

Single Stranded Conformational Polymorphism analysis detects SNPs that have changed the conformation and mobility of the DNA via electrophoresis

Question 19

What are the cons of SSCP analysis?

Answer 19

• Requires Ag staining
• Conditions must be perfect
• Difficult to automate
• Time consuming