LESSON 8 POLYMERASE CHAIN REACTION Flashcards
(175 cards)
1
Q
DNA extraction → PCR→
A
Gel electrophoresis → DNA sequencing
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)
3
Q
Invented by
A
Dr. Kary Mullis in 1983
4
Q
Dr. Kary Mullis in 1983 was working in
A
Cetus Corporation California
5
Q
Won Nobel Prize for Chemistry in
A
1995
6
Q
He is trying to find other purpose for the companies’ [?] (primers: 16s rRNA)
A
oligonucleotides
7
Q
: machine used in PCR
A
Thermocycler
8
Q
: use din academic level
A
Conventional PCR
9
Q
: shows the amount of target gene amplified; provides more information; no need for gel electrophoresis
A
RT-PCR
10
Q
Discovery of double-helix structure of DNA
A
1953
11
Q
Identification of first DNA polymerase
A
1957
12
Q
Invention of DNA repair synthesis (single primer system)
A
1971
13
Q
Proposal of two primer system
A
1971
14
Q
Incorporation of Taq-polymerase into PCR
A
1988
15
Q
Invention of PCR
A
1983
16
Q
Amplify specific segments of DNA by enzymatic method (using Taq polymerases) and cycling condition (cycle of temperatures: 95, 55, 72oC)
A
PCR
17
Q
: use of water bath and transferring manually
A
Traditional PCR
18
Q
: changing of temp continuously
A
Conventional thermocyler
19
Q
PCR can be further used in:
A
Gene sequencing
Screening of genetic abnormalities Eg. Factor V Leiden Mutation
Diagnosis of infectious diseases
20
Q
: once amplified, genetic code can be revealed
A
Gene sequencing
21
Q
for identification of new bacte spp
A
Gene sequencing
22
Q
used in the clinical setting to determine mutations
A
Screening of genetic abnormalities Eg. Factor V Leiden Mutation
23
Q
one of the most common hereditary thrombosis (clot formation)
A
Screening of genetic abnormalities Eg. Factor V Leiden Mutation
24
Q
: new/emerging diseases
A
Diagnosis of infectious diseases
25
Could PCR detect antigens and antibodies?
No
26
Are RBCs suitable specimen for PCR?
No
27
: only the gene that encodes and Ag or Ab can be determined
PCR is a nucleic acid test
28
Detects genetic materials only in [?]: can only detect plasmids, viruses
nucleocapsids/ nucleus
29
In-vivo: Replicates an entire DNA strand
REPLICATION
30
In-vitro: Replicates only a segment of DNA strand
PCR
31
RNA primers
REPLICATION
32
DNA primers
PCR
33
Helicase
REPLICATION
34
Heating
PCR
35
DNA polymerase
REPLICATION
36
Taq Polymerase
PCR
37
Continuous process
REPLICATION
38
Discontinuous process: 20-40 Cycles
PCR
39
Less error and faster
REPLICATION
40
More error and slower
PCR
41
1st cycle
2^1 = 2 copies
42
2nd cycle
2^2 = 4 copies
43
3rd cycle
2^3 = 8 copies
44
30th cycle
2^30 = 9 copies
45
• 94-98°C for 1-3 minutes
Denaturation
46
• breaks the hydrogen bonds between the two strands of DNA and converts it into a single-stranded DNA.
Denaturation
47
• The single strands now act as a template for the production of new strands of DNA
Denaturation
48
• 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.
Annealing
49
• primers bind to their complementary sequences on the template DNA.
Annealing
50
• Then DNA polymerase then binds to the template-primer hybrid and starts the DNA synthesis.
Annealing
51
• 72-80°C for about 2 minutes
Elongation and Extension
52
• bases are added to the 3' end of the primer by the Taq polymerase enzyme.
Elongation and Extension
53
• This elongates the DNA in the 5' to 3' direction.
Elongation and Extension
54
• 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.
Elongation and Extension
55
Separation of DNA strands
Denaturation
56
: template DNA is separated by H bonds
At 95°C
57
: resistant to very high temp
Taq polymerase
58
: destroyed at >37°C
DNA polymerase
59
marks the spot/area/target gene where it will be amplified
Primers
60
: building blocks to create a new strand
Nucleotides
61
Thermocycler is set at [?] (multiplies 35 times; 235)
35 cycles
62
: amplifying/photocopying of target gene multiple times
PCR
63
3 imp reagents:
primers (bookmarks; site where Taq polymerase will extend/elongate the new strand), polymerases, nucleotides
64
Cyclical
65
2 primers attaches to 3’ end:
forward and reverse primers
66
Computed by:
(melting temperature of primers – 5)
67
Ex. Tm = 56.1°C; Annealing temp =
51.1°C
68
attaches first before Taq polymerase
Primer
69
After addition of reverse primer and DNA/Taq polymerase
Elongation and Extension
70
: needed to elongate the new strand
Deoxynucleotide triphosphate (DNTPs)
71
building blocks of the new strand
Deoxynucleotide triphosphate (DNTPs)
72
depends on the number of bases in the primer
Elongation
73
is based on how long the primer is
Time of elongation
74
16s rRNA:
20 seconds
75
Thermocycler:
21 seconds at 72oC
76
Initial denaturation
94°C
2min
77
Denaturation
94°C
45sec
78
Annealing
55°C to 65°C
45sec
79
Extension
72°C
45sec
80
Final extension
72°C
7min
81
: yellow
Taq polymerase
82
: white
DNTPs
83
: maroon and green
Forward and Reverse primers
84
No amplification will take place if one component is missing
TRUE
85
From DNA extraction method, it is the target sequence
DNA template/Target DNA
86
Around: 5-50ng of DNA
DNA template/Target DNA
87
Example of template that we can use:
Genomic DNA (from humans); cDNA
88
Bracket/ bookmark the target sequence on the template
Primers (Forward and Reverse)
89
provide a starting point for replication
Primers (Forward and Reverse)
90
Add nucleotides to the complementary to template
DNA polymerase (Taq polymerase)
91
Construction workers; adds the nucelotides
DNA polymerase (Taq polymerase)
92
Attaches to the target sequence; Dictates which one to amplify
Primers (Forward and Reverse)
93
Extraction must have high purity or it will create poor PCR
DNA template/Target DNA
94
RNA + Reverse Transcriptase(enzyme) =
cDNA
95
: bind with the antisense strand or the noncoding strand or the template strand
Forward primer
96
: bind with the sense strand/non template strand
Reverse primer
97
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.
template strand
complementary strand
no amplification
98
Does PCR use DNA or RNA primers?
PCR uses [?]. [?] are used in replication in vivo.
DNA primers
RNA primers
99
• A primer has to be specifically designed according to the [?] it has to amplify in the PCR technique.
strands
100
• There are numerous tools available now to design a primer:
GenScript, Primer-BLAST
101
• 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 [?].
18 to 24
DNA region
melting temperature
loops
dimers
102
forward primer attaches to the [?]; reverse primer attaches to [?]
antisense strand
sense strand
103
• These primers are used to bind to the
16s rRNA gene
104
is a sequence of DNA encoding the RNA of the small subunit of the ribosome of bacteria.
• 16s rRNA gene
105
• seen in all the bacteria hence it became a tool to identify and detect pathogenic bacteria through PCR
• 16s rRNA gene
106
Universality
• 16s rRNA gene
107
Activity in cellular functions
• 16s rRNA gene
108
Extremely conserved structure and sequence
• 16s rRNA gene
109
Considered to be the standard method to identify, and for taxonomic classification of bacterial species
16s gene sequencing
110
Describing new species and novel pathogen which were never cultured successfully in labs
16s gene sequencing
111
Reclassify bacteria into whole new genera or species
16s gene sequencing
112
It's sequencing in microbiology acts as an inexpensive and quick substitute to the phenotypic techniques of identifying bacteria
16s gene sequencing
113
Some regions of these gene sequences render a species-specific signature sequence used in identifying bacteria
16s gene sequencing
114
The nucleotide probes are applied to identify sequence analysis, phylogenetic analysis, clinical bacteria, and bacteria's molecular classification
16s gene sequencing
115
What makes a good DNA Polymerase and why Taq Polymerase is popular?
Highly Specific
High thermostability
Good processivity
Average fidelity (proofreading capacity)
116
Low non specific amplification
Highly Specific
117
Half-life of approximately 40 min at 95°C
High thermostability
118
should not be destroyed in the course of PCR
High thermostability
119
Incorporates nucleotides at a rate of about 60 bases per second at 70°C and can amplify lengths of about 5 kb
Good processivity
120
fast
Good processivity
121
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
Average fidelity (proofreading capacity)
122
less error rate
Average fidelity (proofreading capacity)
123
: very polular source of DNA for PCR
Taq Polymerase (Thermos aquaticus)
124
: produces pfu DNA polymerase
Pyrocuccus furiosus (Pfu)
125
Components of PCR Cont.
Nucleoside triphosphates
Magnesium chloride (MgCl2)
Buffer
126
• as building blocks of new DNA strand
Nucleoside triphosphates
127
• dNTPs (dATP, dCTP, dGTP, dTTP)
Nucleoside triphosphates
128
• stabilizes interaction between primers, template and polymerase and the phosphate group of a dNTP
Magnesium chloride (MgCl2)
129
• cofactor for activity of DNA polymerases by enabling incorporation of dNTPs during polymerization
Magnesium chloride (MgCl2)
130
• The magnesium ions at the enzyme's active site catalyze phosphodiester bond formation between the 3-OH of a primer
Magnesium chloride (MgCl2)
131
improves the attachment of DNA polymerase in the strand
Magnesium chloride (MgCl2)
132
• Regulates pH for optimal enzyme activity at ~ 8.5
Buffer
133
maintains an alkaline environment
Buffer
134
function at the active site of DNA polymerase
Magnesium ion's
135
helps to coordinate interaction between the 3'-OH of a primer and the phosphate group of an incoming dNTP (hollow block) in DNA polymerization.
Mg2+
136
- attaches to polymerase to improve attachment to primer; to stabilize the structure
Magnesium ions
137
Ensuring success in our PCR Test
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
• Sterile filter tips
Sterile environment
139
• Dedicated equipments
Sterile environment
140
• Apply 70% ethanol spray for cleaning microbes, to denature protein and dissolve lipid
Sterile environment
141
• Change your gloves often
Sterile environment
142
• Freezing and thawing the reagents too many times could damage enzymes and dNTPs due to recrystallization
Inventory the aliquoted PCR reagents
143
• Ta = 3-5°C lower than the Melting tempt
Correct annealing temperature (Ta)
144
• Too high Ta will produce insufficient primertemplate hybridization, resulting in low PCR product yield.
Correct annealing temperature (Ta)
145
• Too low Ta may possibly lead to non-specific products, caused by a high number of base pair mismatches
Correct annealing temperature (Ta)
146
• Make sure each reagent is added in proper concentration and is not left out
Check off each reagent as it's added to the master mix
147
• Leaving out a reagent= NO
Check off each reagent as it's added to the master mix
148
• better quality = better PCR result
Check DNA quality
149
Polymerase chain reaction
PCR
150
Quantitative polymerase chain reaction
qPCR
151
Primer amplification
PCR
152
Either probe hydrolysis or fluorescence through intercalating dye
qPCR
153
Non-fluorescence
PCR
154
Fluorescence
qPCR
155
PCR primers, Taq DNA polymerase, PCR buffer and template DNA
PCR
156
Set of probes, dye, primer set, PCR buffer, template DNA, taq or reverse transcriptase enzyme
qPCR
157
Reaction preparation, amplification and agarose gel electrophoresis.
PCR
158
Reaction preparation, amplification and real time detection.
qPCR
159
DNA bands on gel
PCR
160
Peak or graph of amplicons
qPCR
161
Low resolution amplification
PCR
162
High resolution
qPCR
163
Amplification, detection of mutation
PCR
164
Amplification and quantification
qPCR
165
In this type, the DNA amplification is detected in real-time with the help of a fluorescent reporter.
Real-time PCR/ Quantitative qPCR
166
The signal strength of the fluorescent reporter is directly proportional to the number of amplified DNA molecules.
Real-time PCR/ Quantitative qPCR
167
Tells how many molecules were amplified
Real-time PCR/ Quantitative qPCR
168
Ex. Covid-SARS 2: high amplification in the 25th cycle = positive; 2oth = negative
Real-time PCR/ Quantitative qPCR
169
Uses reverse transcription to produce a DNA template from an RNA source
RT-PCR (Reverse transcription PCR)
170
RNA + reverse transcriptase =
complementary DNA (CDNA)
171
This was designed to improve sensitivity and specificity.
Nested PCR
172
They reduce the non-specific binding of products due to the amplification of unexpected primer binding sites.
Nested PCR
173
Provides more specific amplification
Nested PCR
174
This is used for the amplification of multiple targets in a single PCR experiment.
Multiplex PCR
175
It amplifies many different DNA sequences simultaneously
Multiplex PCR
