[LAB] Unit 4.1 PCR

Question 1

2 big issues solved by PCR

Answer 1

Specificity
Amplification

Question 2

The human genome is composed of how many bp?

Answer 2

3.4 B bp

Question 3

E. coli is composed of how many bp?

Answer 3

4.6 M bp

Question 4

T or F: There are a lot of other sequences in a genome that we’re not interested in detecting.

Answer 4

T

Question 5

To be visible on an agarose gel, need around ____ DNA for fluorescent stain (or around 25ng for FastBlast).

Answer 5

10ng

Question 6

Avogadro’s number

Answer 6

6.02e23

Question 7

PCR was developed by _____ ______ in _____

Answer 7

Kary Mullis; mid-1980s

Question 8

Mullis was granted a Nobel prize in chemistry in ____

Answer 8

1993

Question 9

PCR is also known as a “_____ machine” for DNA

Answer 9

copy

Question 10

T or F: The PCR is a simple technique developed in 1985 to amplify sequence-specific DNA fragments in vivo.

Answer 10

F

in vitro

Question 11

T or F: PCR can be performed in as fast as 1 hour

Answer 11

T

Question 12

PCR amplification is achieved by using ________ primers.

Answer 12

oligonucleotide

Question 13

These are typically short, single stranded oligonucleotides which are complementary to the outer regions of known sequence.

Answer 13

Oligonucleotide primers

Question 14

The oligonucleotides serve as primers for DNA polymerase and the denatured strands of the large DNA fragment serves as the ________.

Answer 14

template

Question 15

T or F: The new strands have defined 5’ ends (the 5’ ends of the oligonucleotide primers), whereas the 3’ ends are potentially ambiguous in length.

Answer 15

T

Question 16

T or F: During PCR, the DNA Polymerase is used as a template to synthesize a new DNA strand.

Answer 16

F

the existing DNA molecule

Question 17

This causes strands to separate

Answer 17

Heat

Question 18

Exact-length target product is made in the ___ cycle

Answer 18

3rd

Question 19

T or F: Low [Mg2+] increases specificity

Answer 19

T
↓ Mg2+; ↑ Specificity; ↓ Yield

Question 20

T or F: High [Mg2+] stabilizes primer annealing, can increase sensitivity, can decrease primer specificity

Answer 20

T
↑ Mg2+; ↑ Sensitivity; ↓ Specificity

Question 21

Effect of too little DNA Polymerase

Answer 21

Insufficient product

Question 22

Effect of too much DNA Polymerase

Answer 22

decreased specificity

Question 23

2 primers anneal each other instead to the intended DNA sequence

Answer 23

Primer-dimers

Question 24

Taq Pol activity decreases above ____

Answer 24

93 C

Question 25

Increase number of cycles if rxn has ____ initial target molecules

Answer 25

<103

Question 26

Inhibit amplification of nucleic acids by PCR

Answer 26

PCR Inhibitors

Question 27

Common PCR inhibitors

Answer 27

Heparin
Urea
Hemoglobin

Question 28

A strand of short nucleic acid sequences that serves as a starting point for DNA synthesis

Answer 28

Primer

Question 29

The polymerase starts replication at the ___-end
of the primer, and copies the opposite strand.

Answer 29

3’-end

Question 30

The longer the primer, the more chance that it is ______

Answer 30

unique

Question 31

T or F: The shorter the primer, the higher melting/annealing temperature

Answer 31

F

The longer

Question 32

The Tm of the probe should be ______ higher than that of the primers

Answer 32

5-10°C

Question 33

The presence of G or C bases within the last five bases from the 3’ end of primers

Answer 33

GC Clamp

Question 34

The length of primer has to be at least ___ bases to ensure uniqueness.

Answer 34

15