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Molecular Biology Lecture > 1: Fundamentals of PCR (MIDTERMS) > Flashcards

1: Fundamentals of PCR (MIDTERMS) Flashcards

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1
Q

The process of making/amplifying DNA copies outside of the body/ in vitro

A

Polymerase Chain Reaction (PCR)

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2
Q

Vitro means?

A

Outside of the body

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3
Q

How many kilobase of DNA are amplified in most PCR methods

A

up to 10 kbs

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4
Q

PCR mirrors what mechanism in its procedure?

A

DNA replication

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5
Q

What are the 2 reasons why we perform PCR?

A
  1. DNA fingerprint (for suspect identification)
  2. Disease identification (analysis of diseases)
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6
Q

Enumerate the 3 main steps of PCR

A
  1. Denaturing
  2. Primer Annealing/Binding
  3. Elongation/extension
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7
Q

3 main step of PCR:

→ DNA is subjected at 95ºC for 30 to 60 seconds in order to separate/unwind

→ Mimics the function of helicase in cells

A

Denaturation of DNA

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8
Q

What temperature and how many seconds is the process of denaturation of DNA in PCR?

A

95ºC for 30 to 60 seconds

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9
Q

T or F

Denaturation of DNA is a step in PCR that can be skipped and is not important

A

F (CANT BE SKIPPED since it is important for DNA to be exposed and primers to bind)

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10
Q

3 main step of PCR:

→ The primers bind to their complementary sequences on the single DNA strands

A

Primer annealing/binding

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11
Q

T or F

In primer annealing/binding , primers bind to target sequence and are chosen based on similarand non complementary relationship with target sequence

A

F (primers are chosen based on their COMPLEMENTARY BINDING relationship with target sequence)

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12
Q

What primers exist in PCR?

A

Forward and Reverse primers

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13
Q

Temperatures for primer annealing depends on what?

A

G+C content (guanine and cytosine)

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14
Q

T or F

Higher g+c content, higher melting temperature

A

T

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15
Q
A
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16
Q

Indication of melting temperature of DNA?

A

If half of the strands are in single-stranded state

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17
Q

3 main step of PCR:

→ at 72ºC, DNA polymerase adds nucleotides to the 3’ ends of the primers and is extended

A

Elongation/extension

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18
Q

3 main step of PCR: Elongation

These are building blocks of DNA polymerase

A

dNTPS

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19
Q

What temperature in elongation/extension process of DNA in PCR?

A

72ºC

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20
Q

3 main step of PCR: Elongation

DNA polymerase adds nucleotides where?

A

3’ ends of the primers

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21
Q

3 main step of PCR: Elongation

In what direction does DNA polymerase run along?

A

5’→3’ direction

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22
Q

3 main step of PCR: Elongation

What strand direction favors the polymerase?

A

strand running on 3’→5’

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23
Q

T or F

Extension starts at primers and attaches at appropriate nucleotide

A

T

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24
Q

Final extension cycle runs for how long at what temperature

A

10 minutes at 72ºC followed by incubation at 4ºC

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25
Q

T or F

DNA polymerase typically functions at 80ºC

A

F (37ºC, anything lower or higher could cause it to not perform its function well)

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26
Q

This polymerase used in PCR is a heat stable bacteria purified from hot springs in 1976

A

Thermus aquaticus (Taq DNA polymerase)

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27
Q

T or F

Size of the DNA fragment produced is dependent on size of the polymerase

A

F (dependent on the size of the PRIMER; longer sequence, longer primers)

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28
Q

At each PCR cycle, the number of DNA molecules is increased to how many?

A

Doubled

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29
Q

If you subject your target DNA to 10 cycles, you end up with how many copies of DNA?

A

1, 024 copies of DNA

(2^n lng bb)

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30
Q

If you subject your target DNA to 7 cycles, you end up with how many copies of DNA?

A

128 copies of DNA

(2^n lng bb)

31
Q

T or F

If you start with 2 unwound strands, first cycle produce 4 strands of DNA, While 3rd produces 8

A

T

(basta doubled lang bb)

32
Q

Produced DNA depends on?

A

number of cycles set for thermal cycler

33
Q

Temperature and time for primers annealing/binding?

A

40-65 ºC, 30-60 seconds

34
Q

3 main step of PCR: Primers annealing/binding

T or F

temperature usually set for primers with higher g + c content are usually set 10 ºC-20 ºC below the melting temperature

A

T

35
Q

3 main step of PCR: Primers annealing/binding

Higher g + c content = ?

A

Higher g + c content = higher melting temperature

36
Q

3 main step of PCR: Primers annealing/binding

T or F

the annealing temperature of DNA is where half of the strands are in a single-stranded state

A

F (the MELTING temperature of DNA is where half of the strands are in a single-stranded state

37
Q

thrives in hot temperatures hence it only needs to be added once at the beginning of the procedure

A

Thermus aquaticus Taq DNA polymerase)

38
Q

maximal enzymatic activity of Taq Polymerase?

A

75-80ºC

39
Q

DNA Polymerase functions at what temp?

A

37ºC

40
Q

Enumerate the process of PCR

A
  1. Denaturation
  2. Primer Annealing
  3. Elongation
41
Q

functions ideally at 37ºC

a. DNA Polymerase
b. Taq Polymerase
c. Both
e. NOTA

A

a. DNA Polymerase

42
Q

95ºC for 30 to 60 seconds

a. Annealing
b. Elongation
c. Both
e. NOTA

A

e. NOTA (DENATURATION temp yan)

43
Q

40-65ºC for 30-60 seconds

a. Annealing
b. Elongation
c. Both
e. NOTA

A

a. Annealing

44
Q

72ºC for 10 mins

a. Annealing
b. Elongation
c. Both
e. NOTA

A

b. Elongation

45
Q

mimics function of helicase

a. Elongation
b. Denaturation
c. Both
e. NOTA

A

b. Denaturation

46
Q

requires melting temperature

a. Annealing
b. Elongation
c. Both
e. NOTA

A

a. Annealing

47
Q

95ºC for 5 mins (acc sa table)

a. Initial Denaturation
b. Denaturation
c. Both
d. NOTA

A

a. Initial Denaturation

48
Q

94ºC for 1 min (acc sa table)

a. Initial Denaturation
b. Denaturation
c. Both
d. NOTA

A

b. Denaturation

49
Q

55ºC for 1 min

a. Annealing
b. Denaturation
c. Both
d. NOTA

A

a. Annealing

50
Q

72ºC for 1 min

a. Extension/Extension
b. Final Elongation
c. Both
d. NOTA

A

c. Both

51
Q

4ºC to infinity

a. Annealing
b. Denaturation
c. Both
d. NOTA

A

d. NOTA (hold temp yn bb)

52
Q

What are the essential requirements for PCR?

A
  1. Template dNA
  2. Pair of Primers
  3. Thermostable DNA polymerase
  4. dNTPS
  5. Cations and Buffer
  6. Nuclease-free water
  7. Thermocycler
53
Q

Range of length of primers?

A

15 to 30 nucleotides

54
Q

Essential requirements for PCR:

→ are synthetic oligonucleotides that serve to initiate DNA synthesis

→ short pieces of single-stranded DNA that are complementary to the 2 ends of each target DNA

→ each one provides the 3ʼOH group required to form a covalent bond (phosphodiester) with another nucleotide

A

Primers

55
Q

4 dNTPs used?

A

dTTP, dCTP, dGTP, and dATP

56
Q

recommended concntration of each dNTP?

A

200-250μM

57
Q

T or F

Less than 4mM concentration of dNTP is inhibitory

A

F (MORE THAN 4mM concentration of dNTP is inhibitorykasi magcchelate mg2)

58
Q

are usually supplied with PCR enzymes at an amount that gives a working concentration of 1.5mM when diluted

A

Mg^2+

59
Q

may contaminate and degrade DNA including the primers, template, and amplified product

A

Nuclease

60
Q

T or F

Nuclease-free water must be stored in small aliquots to reduce contamination

A

T

61
Q

a machine that automatically changes the temperatures and incubation times in each cycle

A

Thermocycler

62
Q

Optimal length of primer?

A

18-25 nucleotides

63
Q

15-30 nucleotides

a. Range of primers
b. Optimal length of primers
c. NOTA

A

a. Range of primers

64
Q

18-25 nucleotides

a. Range of primers
b. Optimal length of primers
c. NOTA

A

b. Optimal length of primers

65
Q

long repeats of any base are avoided to minimize?

A

secondary structures

66
Q

T or F

minimize secondary structures by avoiding long repeats of any base as these cause a increase in available primers for cycling

A

F (minimize secondary structures by avoiding long repeats of any base as these cause a DECREASE in available primers for cycling)

67
Q

G+C content should be at what percent?

A

40-65%

68
Q

the final concentration of Mg^2 should be what amount?

A

0.5 to 5mM

69
Q

Higher concentration ofMg^2 = ?

A

Higher concentration ofMg^2 = higher product yield; lower specificity and fidelity

70
Q

Lower Mg^2 concentration =?

A

no PCR Product

71
Q

ideal concentration of dNTPs ?

A

200μM

72
Q

Higher dNTP = ?

A

Higher dNTP = chelates Mg^2–inhibiting DNA polymerase

73
Q

Lower dNTP = ?

A

HIGHER Fidelity; lowver/reduced yield

74
Q
A

Molecular Biology Lecture (17 decks)

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