PCR

Question 1

Components of PCR

Answer 1

target DNA to be amplified is added to a thin-walled tube (Eppendorf) tube and mixed with:
nuclease free water to fill up reaction
primer( backward and forward)
nucleotides (4 dNTPs) building blocks
heat stable DNA polymerase (Taq polymerase)
Buffer and cofactor Mgcl2 (activate Taq)
Thermal cycler

Question 2

Thermal cycler is

Answer 2

heating block that is capable of changing temperature over short intervals

Question 3

PCR cycle

Answer 3

Denaturation: reaction tube heated to approx 94-96 degrees causing separation of target DNA into single strands.
Primer annealing/hybridization: tube is cooled slightly to between 55 and 65 degrees to allow primer to hydrogen bond to the complementary bases at opp ends of target sequence
Primer extension: temp raised to about 70-75 degreed and DNA pol copies target DNA by binding to the 3’ ends of each primer and using primers as templates. DNA pol adds nucleotides to the 3’ end of each primer to synthesize a complementary strand

Question 4

How many times is the cycle repeated

Answer 4

25-40 times and automatically changes temp for each stage. At the end the DNA has been doubled

Question 5

Primer features

Answer 5

short pieces of single-stranded DNA specific for each target. Primers can be specific and random. As primer increases in size the chances of matching the target size increases. The larger the primer the higher the annealing temp

Question 6

Taq DNA pol feature

Answer 6

DNA pol is responsible for synthesis of new DNA strand in 5’-3’ direction
Taq pol is heat stable and has optimal enzymatic activity at 72 degrees. Its enzymatic half-life is at 95 degreed is 40 min .

Question 7

Advantages of PCR

Answer 7

flexible
fast
works on damaged DNA
RNA and DNA can be amplified
High yield amplification can be achieved
PCR products can be directly sequenced
DNA from one cell equivalent can be amplified

Question 8

Disadvantages of PCR

Answer 8

Need info about target DNA sequence
Highly susceptible to contamination or false amplification
Amplification may not be 100% specific
Specificity of amplification is dependent on temp and Mg++ concentration
Analysis and product detection usually take longer that the PCR reaction itself
There is an upper limit to the size of DNA synthesized by PCR