GENETICS - Investigations (PCR, FISH, Linkage, Blotting)

Question 1

What is the optimal temperature for DNA polymerase?

Answer 1

70 degrees

Question 1

What is the optimal length for primers in PCR?

Answer 1

17-20 base pairs

Question 2

Which molecular techniques are mutation detection techniques?

Answer 2

1. DNA sequencing
2. Southern blotting

Question 3

What type of technique is used in Restriction Fragment Length Polymorphisms (RFLPs)?

Answer 3

Southern Blotting

Question 4

What are the steps involved in the cloning process of DNA? (4)

Answer 4

1. Restriction endonucleases cleave sections of human DNA by targeting sugar-phosphate backbone of the DNA.
2. DNA ligase incorporates human DNA into a vector
3. Vector transfers DNA to bacteria
4. Bacteria replicate required segment of DNA

Question 5

What is the difference between southern blotting and northern blotting?

Answer 5

Southern - DNA sequences
Northern - mRNA sequences

Question 6

What is the main limitation of PCR?

Answer 6

High false-positive rate

Question 7

What is the difference between
southern blotting
northern blotting
western blotting
south-western blotting?

Answer 7

southern blotting - detects DNA sequences - treasure map looking SOUTH for DNA treasure

northern blotting- detects mRNA sequences - Northern Rises Up, so detects RNA

western blotting-detects specific proteins - Western cowboys eat protein rich steaks
south-western - DNA-bound proteins (transcription factors)

Question 8

What are the main reagents needed in a PCR? (6)

Answer 8

1. DNA - sequence to be copied
2. Taq polymerase - builds new strands
3. free nucleotides - building blocks
4. Primers - short sequences that adhere to start of target region - tell taq polymerase where to start building from
5. Buffers - maintain pH/concentration of ions for optimal conditions
6. Mg2+ cofactors - primer adheres at correct site and helps taq polymerases to work correctly

Question 9

What are the steps in PCR? (3)

Answer 9

1. Denaturation - boil to 95 degrees to break DNA strands to create two template strands (breaks hydrogen bonds)
2. Annealing - 55 degrees allows primers (18-22 nucleotides in length) to stick to template areas of DNA
3. Extension - 72 degrees - synthesis of complementary DNA by TAQ polymerase synthesising from 5’ to 3’ direction

Each amplification cycle lasts 10 minutes.
Cycle is repeated 50 times, DNA is amplified 105 times.

PCR cycle is able to amplify a single molecule of DNA 10^6 fold in 20 cycles.

Question 10

What is FISH and what is it used for?

Answer 10

FISH is a method of visualising specific locations on a chromosomes - allows detection of chromosomal abnormalities using flourescent DNA probes.

Question 11

What viral vectors are used in DNA recombination?

What non-viral vectors are used in DNA recombination?

Answer 11

Viral: Adenoviruses, Retroviruses, Herpes simplex viruses

Non-viral: liposomes, naked DNA.

Question 12

What are ribozymes used for in gene therapy?

Answer 12

Target to cut the mRNA of the target gene to block transcription.

Question 13

Which test is used to detect microdeletions?

Answer 13

FISH.