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MOLBIO > CHAPTER 6 > Flashcards

CHAPTER 6 Flashcards

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1
Q

transcribe PCR

A

Polymerase Chain Reaction

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2
Q

who invented PCR

A

Dr. Kary Mullis

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3
Q

when was PCR invented

A

1983

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4
Q

where was PCR invented

A

Cetus Corporation California

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5
Q

who won the nobel prize for chemistry in 1995

A

Dr. Kary Mullis

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6
Q

a technique in Molecular Biology used to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence

A

PCR

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7
Q

easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences

A

Polymerase Chain Reaction

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8
Q

Use of PCR

A
  • DNA cloning
  • Diagnosis and monitoring of hereditary diseases
  • amplification of ancient DNA
  • Genetic finger printing and diagnosis of infectious disease
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9
Q

principle of DNA

A

based on the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered DNA

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10
Q

works by enzymatic amplification of DNA making sequence of DNA sequence

A

PCR

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11
Q

needed because DNA polymerase can add a nucleotide only onto a preexisting 3’ - OH group to add the first nucleotide

A

Primer

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12
Q

Components of PCR

A
  • DNA template
  • DNA polymerase
  • Oligonucleotide primers
  • Deoxynucleotide triphosphate
  • Buffer system
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13
Q

The double stranded DNA (dsDNA) of interest, separated from the sample

A

DNA template

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14
Q

Usually a thermostable Taq polymerase that does not rapidly denature at high temperature (98’C), and can function at a temperature optimum of about 70’C

A

DNA polymerase

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15
Q

enzyme that will build new DNA strands

A

DNA polymerase

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16
Q

Short pieces of single stranded DNA which are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence

A

Oligonucleotide primers

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17
Q

Single units of the bases A,T,G, and C provide the energy for polymerization and the building blocks of DNA synthesis

A

Deoxynucleotide Triphosphate

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18
Q

examples/nucleotides of dNTPs (Deoxynucleotide Triphosphate)

A
  • dATP
  • dTTP
  • dGTP
  • dCTP
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19
Q

Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation

A

Buffer system

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20
Q

also important for polymerase activity, stability, and fidelity

A

Buffer system

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21
Q

maintains proper conditions for DNA polymerase

A

Buffer system

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22
Q

how many base pairs are there in a primer

A

20-30 base pairs

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23
Q
A
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24
Q

melting temperature of primers

A

50-60’C

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25
half life of DNA polymerase
40 min at 95'C
26
2 types of DNA polymerase
- Taq polymerase - Pfu (Proof reading) polymerase
27
has fidelity that is 7x that of Taq DNA polymerase but its synthesis rate is less than half that of Taq polymerase
proofreading Pfu DNA polymerase
28
Steps for DNA polymerase
1. Denaturation 2. Annealing 3. ELongation ro Extension
29
This step involves heating the reaction mixture to 94-98'C for 1-3 minutes
Denaturation
30
the double strands DNA is denatured to single strands due to breakage in weak hydrogen bonds
Denaturation
31
temperature for denaturation
94-98'C for 1-3 minutes
32
The reaction temperature is rapidly lowered to 54-60'C for 20-40 seconds
Annealing
33
This stage allows the primers to bind to their complementary sequence in the template DNA
Annealing
34
temp for Annealing
54-60'C for 40-60 seconds
35
also known as extension
Elongation
36
this step usually occurs at 72-80'C (most commonly 72'C)
Elongation
37
In this step, the polymerase enzyme sequentially adds bases to the 3' each primer, extending the DNA sequence in the 5' to 3' direction
ELongation
38
Under optimal conditions, DNA polymerase will add about _________ bp/minute
1000
39
temp for elongation
72-80'C (commonly 72'C)
40
cycles in PCR
35 cycles
41
Ways to ensure successful PCR
1. Sterile environment 2. Inventory of aliquoted PCR reagents 3. Correct annealing temperature 4. Check off each reagent as its added to the master matrix 5. CHeck DNA quality 6. Check magnesium concentration
42
Types of PCR
1. Real-time PCR/ Quantitative PCR 2. Reverse Transcription PCR 3. Multiflex-PCR 4. Nested PCR
43
44
it is used to amplify unknown DNA segment that flanks one end of known DNA sequence for which no primers are available
reverse transcription PCR
45
converts RNA to DNA before amplification
reverse transcription PCR
46
This is used for the amplification of multiple targets in a single PCR experiment
multiflex PCR
47
It amplifies many different DNA sequences simultaneously
multiflex-PCR
48
this was designated to improve sensitivity and specificity
nested PCR
49
they reduce the non-specific binding of products due to the amplification of unexpected primer binding sites
nested PCR
50
Properties of a good DNA polymerase
1. Low nonspecific amplification 2. Half-life of approximately 40 min at 95'C 3. Incorporates nucleotides at a rate of about 60 base per second at 70'C and can amplify lengths of about 5kb 4. proofreading Pfu DNA polymerase has fidelity that is 7x that of Taq DNA polymerase, but its synthesis rate is less than half that of Taq polymerase
51
enzyme for RTPCR
reverse transcriptase
52
what primers are utilized for nested PCR
- 2 forward - 2 reverse
53
multiflex PCR is used for the detection of:
- pathogens - genetic conditions - forensic studies
54
for analyzing low abundance DNA sample
Nested PCR
55
principle is primer amplification
PCR
56
principle is either probe hydrolysis or fluorescence through intercalating dye
qPCR
57
transcribe qPCR
Quantitative Polymerase chain reaction
58
Chemistry: non-fluorescence
PCR
59
Chemistry: Fluorescence
Quantitative polymerase chain reaction
60
Ingredients of PCR
- PCR primers - Taq DNA polymerase - PCR buffer and template DNA
61
Ingredients of qPCR
- set of probes - dye -primer set - PCR buffer - template DNA - Taq or reverse transcriptase enzyme
62
Assay set up for PCR
- Reaction preparation - amplification - agarose gel electrophoresis
63
Assay set up for qPCR
- Reaction preparation - amplification and real time detection
64
end result for PCR
DNA bands on gel
65
end result for qPCR
Peak or graph of amplicons
66
resolution for PCR
low resolution amplification
67
resolution for qPCR
high resolution
68
application for PCR
- amplification - detection of mutation
69
application for qPCR
amplification and quantification
70
how many molecules are yielded in 35 cycles
2^35
71
3' to 5'
antisense strand
72
5' to 3'
sense strand
73
primers that bind to the antisense strand
Forward primer
74
primers that bind to the sense strand
Reverse primer
75
used to synthesize cDNA (complementary DNA) binding to RNA poly A tail
Oligo dT Primer
76
enzymes that binds to oligo dT primer and synthesizes the cDNA by adding dNTPs
Reverse transcriptase

MOLBIO (8 decks)

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