PCR

Question 1

What is the principle of PCR?

3 points

Answer 1

1. Denaturation
   Heat to separate dsDNA to ssDNA
2. Annealing
   Primers are anneal with H bonding
3. Extension
   Taq polymerase then add nucleotides

Question 2

What are the different types of PCR?

Answer 2

Semi-quantitative PCR

Quantitative competitive PCR

Question 3

Degenerate DNA Primers

Answer 3

To determine a consensus sequence derived from various species

AA can be derived from the 3rd letter of each codon (GGC/GGA/GGU/GGG)

Question 4

Inverse PCR

Answer 4

To clone flanking known sequences
(Unknown-known-unknown)

Light digestion with RE > Ligation > Light digestion with RE > PCR (known-Unknown-known)

Question 5

TA cloning

Answer 5

Where by the terminal transferase activity of Taq DNA polymerase adds 3’ A overhangs.

TA cloning vector will supply it with 3’T overhangs.

Results in high efficiency ligation

Question 6

RAPD

Answer 6

Random amplified Polymorphic DNA

Comparison of the genetic relations between 2 individual

Individual #1
Isolate the DNA> Denature > add random primers> Amplify with PCR > load PCR products into the gel

Individual #2
isolate the DNA> Load PCR products into the gel

Then compare the two results.

Question 7

RT PCR

Answer 7

1) mRNA > dsDNA
Denature > Anneal with Oligonucleotide Primers >extension with RT adds nucleotides

2) use to determine if mRNA corresponds to the gene is present
(Gene expressed > presence of mRNA > RT PCR > DNA copies generated

Question 8

Advantages and disadvantage or RT PCR

Answer 8

Adv: have a full length of cDNA from poly A tail mRNA

Disadvantages: only able to amplify genes with poly A tail

Question 9

Differential display PCR

Answer 9

Simultaneous measurement of expression of many different mRNA molecules

Gene expressed > Presence of mRNA > RT PCR > DNA copies generated

Question 10

RACE

Rapid Amplification of cDNA Ends

Answer 10

Used to isolate the 5’ or 3’ cDNA ends that is in complete

3’ ends
Anchor sequence of Oligio dT at 5’ end > RT > synthesize 2nd strand with internal primer > PCR > many copies

5’ ends
RT with internal primers > terminal transferase and dATP > synthesize 2nd strand with anchor primer > PCR> many copies

Question 11

Direct Mutagenesis

Answer 11

1. Gene in plasmid with mutation target site x
2. Thermal Denaturation (Annealing mutagenic primers-introduce restrictions site)
3. Taq DNA polymerase -many cycles of PCR
4. Transform E.coli cells; screen single colonies for plasmid with unique restriction sites (mutant genes)