Unit 2: Polymerase Chain Reaction

Question 1

template DNA

Answer 1

• section of double stranded DNA (even an entire genome) that contains the section of interest and flanking regions on both sides for primers

Question 2

DNA primers

Answer 2

• oligonucleotide primers (oligo refers to 15-20 bases of single stranded DNA
• prime the synthesis of a new DNA strand and are anti-parallel and complementary to a sequence in the template stand, usually just outside the desired section

Question 3

dNTPs

Answer 3

• the four triphosphate monomers for A, T, G, and C

- dNTP stands for deoxynucleotide triphosphate (dATP, dTTP, dCTP, dGTP)

Question 4

why are the dNTPs considered to be “activated”

Answer 4

• phosphates can be used as the energy source for the replication reaction

Question 5

PCR

Answer 5

• allows us to copy any DNA we want in vitro (in a test tube)
• a specific section of DNA is copied (replicated) over and over which is referred to as “amplifying” the DNA section

Question 6

DNA polymerase (3)

Answer 6

• needed to carry out replication of DNA during PCR
• must be heat tolerant as PCR runs at temperatures between 55-95C
• typical DNA polymerase used is Taq Polymerase, isolated from bacteria that live in hot springs and works best at 75-85C

Question 7

DNA polymerase function

Answer 7

• large enzyme complex responsible for DNA replication in cells and uses the sequence in the template DNA strand to add complementary sequence of deoxyribonucleotides on the newly synthesized strand

Question 8

In what direction does DNA polymerase read DNA

Answer 8

• reads DNA in the 3’ to 5’ direction and makes new DNA in a 5’ to 3’ direction (DNA elongation)

Question 9

What must be present for DNA polymerase to start replicating DNA

Answer 9

• it ABSOLUTELY needs a primer with a free 3’ OH group to start building a new strand of DNA

Question 10

DNA melting

Answer 10

• when DNA is heated it eventually separates/DENATURES/melts into 2 strands

Question 11

If temperature of a hot DNA is slowly lowered, what happens to the DNA?

Answer 11

• DNA will renature (or ANNEAL)

Question 12

What are the three steps in PCR Amplification Cycles

Answer 12

• Denaturation, Anneling, Extension

Question 13

What is the denaturation step?

Answer 13

• A solution containing a double stranded DNA (the template duplex) is heated to separate the DNA into 2 individual strands (about 95C)

Question 14

What is the annealing step

Answer 14

• when the solution is cooled, the 2 primers anneal to their complementary sequence on the strands of the template duplex (about 55-60C)

Question 15

What is the extension step?

Answer 15

• DNA polymerase synthesizes new DNA strands (complementary to the template duplex strands) by extending primers in a 5’ to 3’ direction

Question 16

how long are primers and how are the oriented on the DNA template?

Answer 16

• typically 20-30 nucleotides long and are oriented with their 3’ ends toward each other

Question 17

How do we ensure pairing of primers with complementary sequences?

Answer 17

• primers are added to the reaction mixture in great excess

Question 18

After each round of amplification, how many molecules have the same sequence as the template duplex?

Answer 18

• double the amount

Question 19

After n cycles of amplification, how many copies of the target sequence are there?

Answer 19

• 2^n copies