U4 LAB: POLYMERASE CHAIN REACTION

Question 1

Two big issues in Identification of a Specific Sequence

Answer 1

• Specificity
• Amplification

Question 2

E. coli

Answer 2

4.6 million bp

Question 3

HIV

Answer 3

9.7 thousand bp

Question 4

Ability to detect the target DNA strand to a group of various DNA strands

Answer 4

Specificity

Question 5

Avogadro’s number

Answer 5

6.02^23

Question 6

PCR was developed by?

Answer 6

Kary Mullis in mid 1980s

Question 7

Amplification is done?

Answer 7

exponentially

every product will be used for the next cycle

Question 8

A relatively simple technique developed in 1985 to amplify sequence-specific DNA fragments in vitro

Answer 8

Polymerase Chain Reaction (PCR)

Question 9

This is an enzymatic process in which a target DNA sequence is copied by?

Answer 9

DNA polymerase

Question 10

PCR amplification is achieved by using?

Answer 10

short, single stranded, oligonucleotide primers

Question 11

Serve as primers for DNA polymerase

Answer 11

oligonucleotides

Question 12

Serves as template

Answer 12

Denatured strands of the large DNA fragment

Question 13

The shorter the primer, the ________ the process

Answer 13

faster

Question 14

T/F: PCR can amplify a specific subsequence out of a much larger DNA.

Answer 14

True

Question 15

T/F: Each PCR cycle theoretically doubles the amount of DNA.

Answer 15

True

Question 16

Components for PCR Amplification

Answer 16

• PCR
• Template DNA
• Target specific forward and reverse oligonucleotide primers
• PCR buffer with MgCl2
• Each of the 4 dNTPs
• Taq polymerase

Question 17

PCR buffer is mixed with?

Answer 17

MgCl2 (cofactor for DNA polymerase)

Question 18

under dNTPs

Answer 18

dATP, dCTP, dGTP, dTTP

Question 19

This refers to the thermostable DNA polymerase.

Answer 19

Taq polymerase

Question 20

Temp for Primer Annealing

Answer 20

50 to 65

30 is seldom used

Question 21

Temp for Denaturation

Answer 21

94-95

Question 22

Temp for Primer Extension

Answer 22

72

Question 23

As temperature is raised, the DNA strands?

Answer 23

separate or denature

Question 24

The temperature is then lowered to?

Answer 24

allow primers to attach or anneal

Question 25

The temperature is raised after annealing to provide optimum environment for a special heat resistant type of DNA polymerase called?

Answer 25

Taq polymerase

Question 26

Taq polymerase stands for?

Answer 26

Thermus aquaticus

Question 27

After five cycles, _______ copes have been produced.

Answer 27

32

Question 28

Taq polymerase binds to?

Answer 28

double-stranded substrate

Question 29

This is a perfected version in which there is no more excess after cycles, only contains the target DNA sequence.

Answer 29

amplicons

Question 30

This effect happens in the absence of a primer.

Answer 30

Plateau effect

Question 31

Affinity to detect DNA target sequence if you have a large amount of DNA sample

Answer 31

Specificity

Question 32

Ability to detect target sequence if you have a small amount of DNA sample

Answer 32

Sensitivity

Question 33

Low Mg2+

Answer 33

increase specificity, decreased yield

Question 34

High Mg2+

Answer 34

increase sensitvity, decrease specificity

Question 35

Too much DNA polymerase?

Answer 35

decreases specificity

Question 36

Too little DNA polymerase?

Answer 36

insufficient product

Question 37

Pfu polymerase stands for?

Answer 37

Pyrococcus furiosus

Question 38

Antibody binds and inactivates Taq polymerase at?

Answer 38

room temperature

Question 39

Polymerase is activated when?

Answer 39

heat denatures and releases antibody

Question 40

This technique prevents formation of primer-dimers and other non-specific products

Answer 40

Hot Start Techniques

Question 41

This is when 2 primers anneal each other instead to the intended DNA sequence.

Answer 41

Primer-dimers

Question 42

Denaturation temp

High can?

Answer 42

increase sensitvity

Question 43

Denaturation temp

Taq pol activity can?

Answer 43

decrease above 93C

Question 44

Primer annealing temp

Increase in temp?

Answer 44

increases specificity

Question 45

Primer annealing temp

Decrease in temp?

Answer 45

increases sensitivity

Question 46

Duration time of primer extension

Longer times increase?

Answer 46

sensitivity

Question 47

Cycle number

_____ number of cycles if rxn has _____ initial target molecules

Answer 47

Increase, <103

Question 48

PCR inhibitors interact?

Answer 48

directly with DNA or interfere with DNA polymerases

Question 49

PCR inhibitors are endogenous to sample or introduced during?

Answer 49

sample processing or DNA purification

Question 50

Common inhibitors

Answer 50

• Hemoglobin
• Urea
• Heparin