DNA Markers

Question 1

RFLPS- Southern blotting

Answer 1

• Restriction Fragments Length Polymorphisms
• Digestion of genomic DNA with restriction enzymes, result will be a smear.
• To target the smear we do a southern blot.

Question 2

PCR- RFLPS

Answer 2

• Amplification of a specific region by PCR.
• Digestion with restriction enzymes after PCR.
• Agarose Gel Electrophoresis to see the result.

Question 3

What is the blurred band on a DNA ladder?

Answer 3

Primer Dimers

None specific banding- primers anneal to eachother and DNA polymerase starts adding nucleotides to 3 prime end of primer.

Question 4

VNTRs- DNA Fingerprinting

Answer 4

• Extract DNa
• Quantify DNA
• Digest DNA with restriction enzyme.
• Run on agarose gel.
• Souther blot to transfer DNA to membrane.
• Probe membrane with multi to single locus probe.
• Detect bound probe by autoradiography

Question 5

STRs

Answer 5

• STRS amplified using PCR
• Agarose Gel Electrophoresis does not provide adequate resolution for separation of STRs.
• PAGE provides higher resolution due to smaller pore size.
• PAGE can resolve molecules that differ by a single nucleotide.
• Electrophoresis is carried out under conditions where the two strands stay apart- denaturing conditions.
• To ensure denaturation- heat throughout the whole electrophoresis.
• Fluorescence based detection used.

Question 6

Advantages of capillary electrophoresis over slab electrophoresis.

Answer 6

• Injection, separation and detection can be automated.
• Minute amounts of sample required.
• Short separation time (minutes rather than hours)
• Data available in electronic format.

Question 7

SNPS

Answer 7

• RFLP
• DNA microarrays
• Single base extension

Question 8

Sex Identification

Answer 8

• Amelogenin Gene present on sex chromosomes.
  Primers flanked a 6bp deletion within intron 1 of the gene on the X chromosome.