historical techniques

Question 1

Who pioneered the original method for DNA typing, using what and when

Answer 1

Sir Prof Alec Jefferies in 1984 using VNTRs

Question 2

what is a VNTR

Answer 2

variable number of tandem repeats - also known as minisatellites

Question 3

what is the difference between STRs and VNTRs

Answer 3

the length of the repeating element - can range from several to hundreds of base pairs long

Question 4

how are VNTRs cleaved

Answer 4

using restriction enzymes

Question 5

what are the 6 main stages in DNA fingerprinting (VNTR profiling)

Answer 5

1. DNA extraction
2. Band detection on agarose
3. VNTR Typing via restriction fragment length polymorphism (RFLP)
4. Size separation using gel and Southern transfer
5. Visual comparison
6. Match declared with statistical support

Question 6

What do restriction endonucleases do during RFLP

Answer 6

‘cut’ DNA at different sites to produce fragments

Question 7

why did labs only use one enzyme to cleave during RFLP

Answer 7

insufficient biological material for multiple enzymes

Question 8

why was a second visualisation (Southern Transfer) needed after agarose gel

Answer 8

too many fragments to interpret an individual profile

Question 9

Name 5 factors affecting RFLP

Answer 9

1. DNA degradation - RFLP cleaves high molecular weight DNA and degraded DNA will provide fewer restriction sites
2. Partial RFLP digestion - leads to inconsistent banding
3. STAR activity and point mutations - deviation in the specificity of a cleavage site
4. Electrophoresis artefacts
5. Lack of scientific consensus was exposed - legal teams used poor quality as legal defence

Question 10

Give 3 examples of electrophoresis artefacts that could arise during RFLP

Answer 10

1. Bands can run off gel and patterns can be lost
2. Poor resolution meant that two alleles a single VNTR apart could appear as a single band
3. Band shifting could cause the same alleles to appear two different sizes

Question 11

What does RFLP stand for

Answer 11

Restriction Fragment Length Polymorphism

Question 12

What does AFLP stand for

Answer 12

Amplified Fragment Length Polymorphism

Question 13

how does AFLP differ from RFLP

Answer 13

uses PCR primers to amplify short length VNTRs

Question 14

Name the 2 benefits of AFLP before STRs became the dominant marker

Answer 14

1. Improved sensitivity compared to RFLP
2. Worked on degraded samples