Lab 9

Question 1

what is a polymerase chain reaction (PCR)?

Answer 1

a method used widely in molecular biology to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail

Question 2

What is PCR used for?

Answer 2

to amplify the DNA sequences

Question 3

Denaturing step

Answer 3

94 degree Celsius
30 sec
*Denatures the target DNA

Question 4

Annealing step

Answer 4

30-68 degree Celsius
30 secs
*anneals the primers

Question 5

Extension Step (elongation)

Answer 5

65-75 degree Celsius (72)
0.5-3 min
*DNA strand elongate
**Cycle repeated 30+ times

Question 6

what is polymorphism?

Answer 6

a discontinuous genetic variation resulting in the occurrence of several different forms or types of individuals among the members of a single species

Question 7

Genetic polymorphism is used to study what?

Answer 7

Human evolution
Identity and disease testing

Question 8

What is a Alu?

Answer 8

a transposon or transposable element, it’s a member of the family of short interspersed elements (SINEs)

Question 9

Approximately how long is Alu?

Answer 9

300-bp in length

Question 10

Where is the restriction enzyme Alu I found?

Answer 10

located near the middle of Alu element

Question 11

where is Alu transposons only found?

Answer 11

Primate genomes

Question 12

Human chromosome has more than how many million copies of Alu?

Answer 12

one million- about 10% of the genome by mass

Question 13

the Alu insertion may be present or absent on each of the paired chromosomes—this insertion is

Answer 13

dimorphic, meaning it is present in some individual and not in others

Question 14

PV92 polymorphism is phenotypically

Answer 14

neutral—it has no known relationship to any trait or disease state

Question 15

Overall, PCR is a technique for

Answer 15

generating large quantities of a specified DNA

Question 16

PCR is a cell free amplification technique for

Answer 16

synthesizing multiple identical copies of any DNA of interest

Question 17

what are the requirements for PCR?

Answer 17

*A target for DNA (100-35000 bp in length)
*Two primers (17-30 nucleotides)
* dATP, dCTP, dGTP, dTTP
* dna polymerase that can withstand temp at 95 degrees celsius

Question 18

Denaturation

Answer 18

Increasing the temp to 95 degrees celsius for 1 min, the DNA gets denatured and two strands separate

Question 19

Renaturation or annealing

Answer 19

As the temp of mixture is slowly cooled to about 55 degrees celsius, the primers base pair with complementary regions flanking target DNA strands

Question 20

Synthesis

Answer 20

*initiation of DNA synthesis occurs at 3” hydroxyl end of each primer
*the primers are extended by joining the bases complementary to DNA strands

Question 21

what is the optimum temperature for DNA polymerase?

Answer 21

75 degrees celsius

Question 22

What is the optimum temp of E.coli DNA polymerase?

Answer 22

37 degrees celsius

Question 23

How can u stop a reaction?

Answer 23

by raising the temp to 95 degrees Celsius

Question 24

How long does each PCR take about?

Answer 24

3-5 min

Question 25

Cycle 1

Answer 25

• new dna strand is joined to each primer is beyond the sequence that is complementary to second primer
  -new strands are referred as long template
  -will be used in the second cycle

Question 26

Cycle- 2

Answer 26

• the DNA strands are denatured, annealed with primers and subjected to DNA synthesis

-at the end of this round, long templates and short templates are formed

Question 27

Cycle 3

Answer 27

• the original DNA strands along with long and short templates are starting materials
  -Denaturation, renaturation and synthesis are repeated again and again for each cycle
  -at the end of the 32nd cycle of PCR, about 1 million fold target DNA is synthesized

Question 28

what fragment of E. coli DNA polymerase is used in original technique?

Answer 28

Klenow, gets denatured at higher temperature, therefore, fresh enzyme had to be added for each cycle

Question 29

Taq DNA polymerase is what?

Answer 29

heat resistant, not necessary to freshly add this enzyme for each cycle