historical techniques:

Question 1

origins of DNA typing:

Answer 1

Method pioneered by Jefferies in 1984.
Applied by Gill at Forensic Science Service.
Used VNTR markers.
-Variable Number of Tandem Repeats. Also known as ‘minisatellites’.
Used in research and casework until early 1990’s.
Like STRs, VNTRs are comprised of repeating units.
The difference is the length of the repeating element.
-Repeating unit can range from several to 100s of base pairs long. Therefore, the fragment size can be kilobases (thousands) long.
A number of different VNTRs were used simultaneously. These were cleaved using restriction enzymes.
The banding pattern generated were termed ‘DNA fingerprints’.

Question 2

PCR:

Answer 2

Around the same time Mullis and Faloona invent PCR.
-PCR not routinely used in forensic cases until later.
-Obvious limitations around sensitivity.
By early 1990’s STRs were becoming more popular.
-Not routinely used in forensic cases until later.

Question 3

DNA fingerprinting:

Answer 3

DNA fingerprinting is a multi step process:
-DNA extraction, band detection on agarose, VNTR typing via restriction fragment polymorphism (RFLP), size separation using gel and southern transfer, visual comparison, match declared with stat support.

Question 4

DNA fingerprinting- RFLP:

Answer 4

VNTR Profiling uses RFLP.
-Restriction Fragment Length Polymorphism.
-Uses restriction endonucleases.
Restriction endonucleases ‘cut’ DNA.
-Different endonucleases cut at different sites.
-Results are many different fragment sizes.
Exist as Type I, II and III.
-Type II used in molecular biology applications.
-Type I and III cut at sites far from recognition sites.

Question 5

DNA fingerprinting- enzymes:

Answer 5

Enzymes chosen to cut at flanking regions
-Labs tended to use a single enzyme to cleave.
-Insufficient biological material for multiple enzymes.
Use of the same enzyme allowed comparison.
-Between samples.
-Between laboratories.
Digested DNA run on an agarose gel.
-Saw cut fragments across whole genome.
-Too many fragments to interpret individual profile.

Question 6

DNA fingerprinting- secondary visualisation:

Answer 6

Needed to undergo secondary visualisation.
-Southern Transfer (Southern Blotting).
-Transferred DNA from gel onto solid matrix.
DNA on agarose gel first denatured.
-Under alkaline conditions (treatment with NaOH).
-Results in single stranded (ssDNA).
ssDNA then adsorbed onto nylon membrane.
-Immobilised by UV cross linking.
-Ready for Single Locus Probe or Multi-Locus Probe analysis.

Question 7

DNA fingerprinting- mlp, slp:

Answer 7

Multi-Locus Probe (MLP) Technique.
-As single probe was used to visualise the VNTRs.
-Probe binds to GC rich region common in VNTR loci.
Mechanism developed by Jeffreys.
-Many loci detected.
-Problem in interpreting mixed DNA profiles.

Single-Locus Probe (SLP) Technique.
-Probe designed to bind to individual VNTR locus.
-DNA profile a lot easier to interpret but less unique.

Question 8

factors affecting RFLP:

Answer 8

DNA Degradation is an issue for VNTRs.
-RFLP cleaves high molecular weight DNA.
-Fewer restriction sites as DNA degrades.
-DNA yield was estimated on gels before RFLP.
Partial RFLP digestion.
-Partial digestion led to inconsistent banding.
-Misidentification of samples as ‘mixed’.
-Check level of digestion with another gel.
STAR activity and point mutations.
-Deviation in the specificity of a cleavage site.

Question 9

factors affecting RFLP- electrophoresis artifacts:

Answer 9

Electrophoresis artifacts.
-Bands can run off gel and patterns can be lost.
-Prevented by using long gels and strict run times.
-Poor resolution meant that 2 alleles a single VNTR apart could be confused as a single band.
-Band shifting could cause the same alleles to appear as 2 different sizes.
-Alleles could adhere to nylon membrane and be visible on next run.
Lack of scientific consensus exposed.
-Legal teams used poor quality as legal defence.

Question 10

AFLP:

Answer 10

AFLP provided an intermediate method between VNTR and STR analysis:
-Amplified Fragment Length Polymorphism.
-Uses PCR primers to amplify short length VNTRs.
-Improved sensitivity – works with degraded samples.
-Commonly used for sex identification using amelogenin.
By late 1990’s STRs became dominant marker detected via capillary electrophoresis.