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Molecular Biology Lec > LESSON 8 POLYMERASE CHAIN REACTION > Flashcards

LESSON 8 POLYMERASE CHAIN REACTION Flashcards

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1
Q

DNA extraction → PCR→

A

Gel electrophoresis → DNA sequencing

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2
Q

Basic set-up:

A

 Containers for pipette tips, reagents (from freezer/on ice), mmicropipette with filtered tips
 Sterile (spray-bottle with 70% ethanol to dissolve proteins, lipids, and unnecessary DNA)

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3
Q

Invented by

A

Dr. Kary Mullis in 1983

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4
Q

Dr. Kary Mullis in 1983 was working in

A

Cetus Corporation California

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5
Q

Won Nobel Prize for Chemistry in

A

1995

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6
Q

He is trying to find other purpose for the companies’ [?] (primers: 16s rRNA)

A

oligonucleotides

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7
Q

: machine used in PCR

A

Thermocycler

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8
Q

: use din academic level

A

Conventional PCR

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9
Q

: shows the amount of target gene amplified; provides more information; no need for gel electrophoresis

A

RT-PCR

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10
Q

Discovery of double-helix structure of DNA

A

1953

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11
Q

Identification of first DNA polymerase

A

1957

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12
Q

Invention of DNA repair synthesis (single primer system)

A

1971

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13
Q

Proposal of two primer system

A

1971

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14
Q

Incorporation of Taq-polymerase into PCR

A

1988

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15
Q

Invention of PCR

A

1983

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16
Q

Amplify specific segments of DNA by enzymatic method (using Taq polymerases) and cycling condition (cycle of temperatures: 95, 55, 72oC)

A

PCR

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17
Q

: use of water bath and transferring manually

A

Traditional PCR

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18
Q

: changing of temp continuously

A

Conventional thermocyler

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19
Q

PCR can be further used in:

A

Gene sequencing

Screening of genetic abnormalities Eg. Factor V Leiden Mutation

Diagnosis of infectious diseases

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20
Q

: once amplified, genetic code can be revealed

A

Gene sequencing

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21
Q

for identification of new bacte spp

A

Gene sequencing

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22
Q

used in the clinical setting to determine mutations

A

Screening of genetic abnormalities Eg. Factor V Leiden Mutation

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23
Q

one of the most common hereditary thrombosis (clot formation)

A

Screening of genetic abnormalities Eg. Factor V Leiden Mutation

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24
Q

: new/emerging diseases

A

Diagnosis of infectious diseases

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25
Q

Could PCR detect antigens and antibodies?

A

No

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26
Q

Are RBCs suitable specimen for PCR?

A

No

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27
Q

: only the gene that encodes and Ag or Ab can be determined

A

PCR is a nucleic acid test

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28
Q

Detects genetic materials only in [?]: can only detect plasmids, viruses

A

nucleocapsids/ nucleus

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29
Q

In-vivo: Replicates an entire DNA strand

A

REPLICATION

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30
Q

In-vitro: Replicates only a segment of DNA strand

A

PCR

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31
Q

RNA primers

A

REPLICATION

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32
Q

DNA primers

A

PCR

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33
Q

Helicase

A

REPLICATION

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34
Q

Heating

A

PCR

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35
Q

DNA polymerase

A

REPLICATION

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36
Q

Taq Polymerase

A

PCR

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37
Q

Continuous process

A

REPLICATION

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38
Q

Discontinuous process: 20-40 Cycles

A

PCR

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39
Q

Less error and faster

A

REPLICATION

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40
Q

More error and slower

A

PCR

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41
Q

1st cycle

A

2^1 = 2 copies

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42
Q

2nd cycle

A

2^2 = 4 copies

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43
Q

3rd cycle

A

2^3 = 8 copies

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44
Q

30th cycle

A

2^30 = 9 copies

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45
Q

• 94-98°C for 1-3 minutes

A

Denaturation

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46
Q

• breaks the hydrogen bonds between the two strands of DNA and converts it into a single-stranded DNA.

A

Denaturation

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47
Q

• The single strands now act as a template for the production of new strands of DNA

A

Denaturation

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48
Q

• The temperature is lowered to approximately 5 °C below the melting temperature (Tm) of the primers (often 54-60°C) to promote primer binding to the template.

A

Annealing

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49
Q

• primers bind to their complementary sequences on the template DNA.

A

Annealing

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50
Q

• Then DNA polymerase then binds to the template-primer hybrid and starts the DNA synthesis.

A

Annealing

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51
Q

• 72-80°C for about 2 minutes

A

Elongation and Extension

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52
Q

• bases are added to the 3’ end of the primer by the Taq polymerase enzyme.

A

Elongation and Extension

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53
Q

• This elongates the DNA in the 5’ to 3’ direction.

A

Elongation and Extension

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54
Q

• Taq Polymerase can tolerate very high temperatures. It attaches to the primer and adds DNA bases to the single strand. As a result, a double-stranded DNA molecule is obtained.

A

Elongation and Extension

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55
Q

Separation of DNA strands

A

Denaturation

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56
Q

: template DNA is separated by H bonds

A

At 95°C

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57
Q

: resistant to very high temp

A

Taq polymerase

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58
Q

: destroyed at >37°C

A

DNA polymerase

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59
Q

marks the spot/area/target gene where it will be amplified

A

Primers

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60
Q

: building blocks to create a new strand

A

Nucleotides

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61
Q

Thermocycler is set at [?] (multiplies 35 times; 235)

A

35 cycles

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62
Q

: amplifying/photocopying of target gene multiple times

A

PCR

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63
Q

3 imp reagents:

A

primers (bookmarks; site where Taq polymerase will extend/elongate the new strand), polymerases, nucleotides

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64
Q

Cyclical

A
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65
Q

2 primers attaches to 3’ end:

A

forward and reverse primers

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66
Q

Computed by:

A

(melting temperature of primers – 5)

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67
Q

Ex. Tm = 56.1°C; Annealing temp =

A

51.1°C

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68
Q

attaches first before Taq polymerase

A

Primer

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69
Q

After addition of reverse primer and DNA/Taq polymerase

A

Elongation and Extension

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70
Q

: needed to elongate the new strand

A

Deoxynucleotide triphosphate (DNTPs)

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71
Q

building blocks of the new strand

A

Deoxynucleotide triphosphate (DNTPs)

72
Q

depends on the number of bases in the primer

A

Elongation

73
Q

is based on how long the primer is

A

Time of elongation

74
Q

16s rRNA:

A

20 seconds

75
Q

Thermocycler:

A

21 seconds at 72oC

76
Q

Initial denaturation

A

94°C
2min

77
Q

Denaturation

A

94°C
45sec

78
Q

Annealing

A

55°C to 65°C
45sec

79
Q

Extension

A

72°C
45sec

80
Q

Final extension

A

72°C
7min

81
Q

: yellow

A

Taq polymerase

82
Q

: white

A

DNTPs

83
Q

: maroon and green

A

Forward and Reverse primers

84
Q

No amplification will take place if one component is missing

A

TRUE

85
Q

From DNA extraction method, it is the target sequence

A

DNA template/Target DNA

86
Q

Around: 5-50ng of DNA

A

DNA template/Target DNA

87
Q

Example of template that we can use:

A

Genomic DNA (from humans); cDNA

88
Q

Bracket/ bookmark the target sequence on the template

A

Primers (Forward and Reverse)

89
Q

provide a starting point for replication

A

Primers (Forward and Reverse)

90
Q

Add nucleotides to the complementary to template

A

DNA polymerase (Taq polymerase)

91
Q

Construction workers; adds the nucelotides

A

DNA polymerase (Taq polymerase)

92
Q

Attaches to the target sequence; Dictates which one to amplify

A

Primers (Forward and Reverse)

93
Q

Extraction must have high purity or it will create poor PCR

A

DNA template/Target DNA

94
Q

RNA + Reverse Transcriptase(enzyme) =

A

cDNA

95
Q

: bind with the antisense strand or the noncoding strand or the template strand

A

Forward primer

96
Q

: bind with the sense strand/non template strand

A

Reverse primer

97
Q

What happens if you only use a forward primer?

If you use only a forward primer, only the [?] will be synthesised but it will not have a [?] to bind to, hence [?] will occur.

A

template strand

complementary strand

no amplification

98
Q

Does PCR use DNA or RNA primers?

PCR uses [?]. [?] are used in replication in vivo.

A

DNA primers

RNA primers

99
Q

• A primer has to be specifically designed according to the [?] it has to amplify in the PCR technique.

A

strands

100
Q

• There are numerous tools available now to design a primer:

A

GenScript, Primer-BLAST

101
Q

• After the design send it to a company that makes primers
1. [?] base pairs in length.
2. It should be specific to the [?] that is to be amplified.
3. The [?] for both the primers should be in a similar range.
4. Highly repeated sequences should be avoided as it can lead to formation of [?] in the primer.
5. The primers designed should be different from each other, otherwise they can anneal to form [?].

A

18 to 24

DNA region

melting temperature

loops

dimers

102
Q

forward primer attaches to the [?]; reverse primer attaches to [?]

A

antisense strand

sense strand

103
Q

• These primers are used to bind to the

A

16s rRNA gene

104
Q

is a sequence of DNA encoding the RNA of the small subunit of the ribosome of bacteria.

A

• 16s rRNA gene

105
Q

• seen in all the bacteria hence it became a tool to identify and detect pathogenic bacteria through PCR

A

• 16s rRNA gene

106
Q

Universality

A

• 16s rRNA gene

107
Q

Activity in cellular functions

A

• 16s rRNA gene

108
Q

Extremely conserved structure and sequence

A

• 16s rRNA gene

109
Q

Considered to be the standard method to identify, and for taxonomic classification of bacterial species

A

16s gene sequencing

110
Q

Describing new species and novel pathogen which were never cultured successfully in labs

A

16s gene sequencing

111
Q

Reclassify bacteria into whole new genera or species

A

16s gene sequencing

112
Q

It’s sequencing in microbiology acts as an inexpensive and quick substitute to the phenotypic techniques of identifying bacteria

A

16s gene sequencing

113
Q

Some regions of these gene sequences render a species-specific signature sequence used in identifying bacteria

A

16s gene sequencing

114
Q

The nucleotide probes are applied to identify sequence analysis, phylogenetic analysis, clinical bacteria, and bacteria’s molecular classification

A

16s gene sequencing

115
Q

What makes a good DNA Polymerase and why Taq Polymerase is popular?

A

Highly Specific

High thermostability

Good processivity

Average fidelity (proofreading capacity)

116
Q

Low non specific amplification

A

Highly Specific

117
Q

Half-life of approximately 40 min at 95°C 

A

High thermostability

118
Q

should not be destroyed in the course of PCR

A

High thermostability

119
Q

Incorporates nucleotides at a rate of about 60 bases per second at 70°C and can amplify lengths of about 5 kb

A

Good processivity

120
Q

fast

A

Good processivity

121
Q

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

A

Average fidelity (proofreading capacity)

122
Q

less error rate

A

Average fidelity (proofreading capacity)

123
Q

: very polular source of DNA for PCR

A

Taq Polymerase (Thermos aquaticus)

124
Q

: produces pfu DNA polymerase

A

Pyrocuccus furiosus (Pfu)

125
Q

Components of PCR Cont.

A

Nucleoside triphosphates

Magnesium chloride (MgCl2)

Buffer

126
Q

• as building blocks of new DNA strand

A

Nucleoside triphosphates

127
Q

• dNTPs (dATP, dCTP, dGTP, dTTP)

A

Nucleoside triphosphates

128
Q

• stabilizes interaction between primers, template and polymerase and the phosphate group of a dNTP

A

Magnesium chloride (MgCl2)

129
Q

• cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerization

A

Magnesium chloride (MgCl2)

130
Q

• The magnesium ions at the enzyme’s active site catalyze phosphodiester bond formation between the 3-OH of a primer

A

Magnesium chloride (MgCl2)

131
Q

improves the attachment of DNA polymerase in the strand

A

Magnesium chloride (MgCl2)

132
Q

• Regulates pH for optimal enzyme activity at ~ 8.5

A

Buffer

133
Q

maintains an alkaline environment

A

Buffer

134
Q

function at the active site of DNA polymerase

A

Magnesium ion’s

135
Q

helps to coordinate interaction between the 3’-OH of a primer and the phosphate group of an incoming dNTP (hollow block) in DNA polymerization.

A

Mg2+

136
Q
  • attaches to polymerase to improve attachment to primer; to stabilize the structure
A

Magnesium ions

137
Q

Ensuring success in our PCR Test

A

Sterile environment

Inventory the aliquoted PCR reagents

Correct annealing temperature (Ta)

Check off each reagent as it’s added to the master mix

Check DNA quality

Check magnesium concentration

138
Q

• Sterile filter tips

A

Sterile environment

139
Q

• Dedicated equipments

A

Sterile environment

140
Q

• Apply 70% ethanol spray for cleaning microbes, to denature protein and dissolve lipid

A

Sterile environment

141
Q

• Change your gloves often

A

Sterile environment

142
Q

• Freezing and thawing the reagents too many times could damage enzymes and dNTPs due to recrystallization

A

Inventory the aliquoted PCR reagents

143
Q

• Ta = 3-5°C lower than the Melting tempt

A

Correct annealing temperature (Ta)

144
Q

• Too high Ta will produce insufficient primertemplate hybridization, resulting in low PCR product yield.

A

Correct annealing temperature (Ta)

145
Q

• Too low Ta may possibly lead to non-specific products, caused by a high number of base pair mismatches

A

Correct annealing temperature (Ta)

146
Q

• Make sure each reagent is added in proper concentration and is not left out

A

Check off each reagent as it’s added to the master mix

147
Q

• Leaving out a reagent= NO

A

Check off each reagent as it’s added to the master mix

148
Q

• better quality = better PCR result

A

Check DNA quality

149
Q

Polymerase chain reaction

A

PCR

150
Q

Quantitative polymerase chain reaction

A

qPCR

151
Q

Primer amplification

A

PCR

152
Q

Either probe hydrolysis or fluorescence through intercalating dye

A

qPCR

153
Q

Non-fluorescence

A

PCR

154
Q

Fluorescence

A

qPCR

155
Q

PCR primers, Taq DNA polymerase, PCR buffer and template DNA

A

PCR

156
Q

Set of probes, dye, primer set, PCR buffer, template DNA, taq or reverse transcriptase enzyme

A

qPCR

157
Q

Reaction preparation, amplification and agarose gel electrophoresis.

A

PCR

158
Q

Reaction preparation, amplification and real time detection.

A

qPCR

159
Q

DNA bands on gel

A

PCR

160
Q

Peak or graph of amplicons

A

qPCR

161
Q

Low resolution amplification

A

PCR

162
Q

High resolution

A

qPCR

163
Q

Amplification, detection of mutation

A

PCR

164
Q

Amplification and quantification

A

qPCR

165
Q

In this type, the DNA amplification is detected in real-time with the help of a fluorescent reporter.

A

Real-time PCR/ Quantitative qPCR

166
Q

The signal strength of the fluorescent reporter is directly proportional to the number of amplified DNA molecules.

A

Real-time PCR/ Quantitative qPCR

167
Q

Tells how many molecules were amplified

A

Real-time PCR/ Quantitative qPCR

168
Q

Ex. Covid-SARS 2: high amplification in the 25th cycle = positive; 2oth = negative

A

Real-time PCR/ Quantitative qPCR

169
Q

Uses reverse transcription to produce a DNA template from an RNA source

A

RT-PCR (Reverse transcription PCR)

170
Q

RNA + reverse transcriptase =

A

complementary DNA (CDNA)

171
Q

This was designed to improve sensitivity and specificity.

A

Nested PCR

172
Q

They reduce the non-specific binding of products due to the amplification of unexpected primer binding sites.

A

Nested PCR

173
Q

Provides more specific amplification

A

Nested PCR

174
Q

This is used for the amplification of multiple targets in a single PCR experiment.

A

Multiplex PCR

175
Q

It amplifies many different DNA sequences simultaneously

A

Multiplex PCR

Molecular Biology Lec (13 decks)

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