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