1. Detection of Variation

Question 1

genotyping vs phenotyping

Answer 1

-genotyping allows us to predict an outcome
-phenotyping allows us to infer a cause

Question 2

steps of work flow

Answer 2

1. decision to determine a genotype/mutation
2. collection of DNA
3. appropriate genotyping method
4. interpretation of results

Question 3

cons of direct DNA sequencing

Answer 3

expensive (equipment, staff), time consuming, not very informative

Question 4

pros of AFLP

Answer 4

simple, cheap, easy interpretation

Question 5

how does AFLP separate fragments?

Answer 5

electrophoresis.

obviously, shorter fragments travel further than longer fragments. DNA dyed with EtBr for visualisation

Question 6

list universal colour system used in direct DNA sequencing

Answer 6

-blue = C
-red = T
-black = G
-green = A

Question 7

pros of RFLP analysis

Answer 7

quick, easy interpretation, cheap

Question 8

cons of RFLP analysis

Answer 8

SNP must be inside RE sites, REs only recognise palindromic sequences

Question 9

describe FRET assay

Answer 9

fluorescence resonance energy transfer
-uses 2 probes which bind to adjacent sites around SNP (and wt) during annealing stage of PCR
-1 probe has fluorescein tag at 3’ end, other probe has LC-red at 5’ end
-probes only fluoresce in close proximity

-if you excite a fluorophore at a wavelength, it emits light at a higher wavelength

Question 10

steps in RFLP genotyping

Answer 10

1. obtain DNA
2. PCR
3. RE digestion
4. electrophoresis

Question 11

pros of fluorescent detection

Answer 11

homogenous closed tube assay
-no post-PCR processing, no tube transfers, RE digestion

quick, simple, streamlined
-reduced labour/reagent cost
-automation potential

decreased potential for sample mix-ups and contamination

Question 12

fluorescent detection cons

Answer 12

requires expertise in molecular biology

probe design is meticulous and expensive

high initial capital outlay (dependent)

Question 13

single nucleotide extension is also known as…

Answer 13

MALDI-TOF MS

(matrix assisted laser desorption/ionisation time-of-flight mass spectrometry)

Question 14

single stranded conformational polymorphism analysis relies on…

Answer 14

fact that if you have a heterozygote position in DNA, and you melt the DNA, you get perfect reannealing

compared to wt DNA, DNA containing a SNP will form a heteroduplex (bubble) and travels differently in acrylamide gel