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Question 1

Polymerase chain reaction (PCR)

Answer 1

amplifies a small amount of DNA into large amounts

Question 2

What is the name of the sample of DNA needing to be copied

Answer 2

The template DNA (often a known sequence or gene)

Question 3

Template DNA

Answer 3

Typically double-stranded and only extremely small quantities are sufficient

Question 4

What are the reagents needed for PCR

Answer 4

• a pair of oligonucleotide primers
• supply of nucleotide triphosphates
• Taq DNA Polymerase (actually makes the copies)

Question 5

What do the oligonucleotide primers have?

Answer 5

sequences complementary to the ends of the template DNA

Question 6

Reverse transcriptase PCR (RT-PCR)

Answer 6

utilizes reverse transcriptase to make a cDNA copy of mRNA. PCR is later used to amplify the cDNA. This is useful as eukaryotic DNA tends to have long stretches of noncoding introns.

Question 7

What are the two steps of RT-PCR?

Answer 7

1. reverse transcriptase recognizes the 3’ end of primers containing repeated thymines and creates a DNA strand complementary to the RNA
2. The RNA strand is replaced with another DNA strand -> double stranded cDNA that can be amplified.

Question 8

What ends of the template strand do the primers anneal to?

Answer 8

5’ end of the sense strand and 3’ end of the antisense strand

Question 9

What is the basic mechanism for PCR?

Answer 9

-denaturation of the template
-annealing of the primers
-making a complementary copy using DNA polymerase

Question 10

Inverse PCR

Answer 10

Method for using PCR to amplify unknown sequences by circularizing the template molecule

Question 11

What does the process of PCR require

Answer 11

changing of temperatures (in a cyclic manner)

Question 12

what is the general sequence of inverse PCR?

Answer 12

DNA is cut with a RE that cuts upstream and downstream but not within it. The linear piece of DNA is circularized and amplified with primers that anneal into the known region. PCR products will have the unknowns that can be cloned.

Question 13

degenerate primers

Answer 13

primer with several alternative bases at certain positions - they are based on amino acid sequences and contain different nucleotides at the wobble position

Question 14

What are the modifications between the PCR cycle and DNA synthesis in vitro

Answer 14

-Double strand template is denatured with high heat (not enzymes)
-the temperature is lowered so primers anneal to their binding sites
-primers bind to opposite strands of the template (one at the beginning and one at the end)

Question 15

What is the most widely used polymerase for PCR?

Answer 15

Taq polymerase (stable and does not denature at high temp.)

Question 16

What is KEY to the PCR process?

Answer 16

The primers must anneal in the correct location

Question 17

What will happen if both primers anneal to the same strand?

Answer 17

The reaction will NOT work