Polymerase Chain Reaction Flashcards
what does PCR produce
millions of copies of a target sequence from template DNA in a few hours, compared with bacterial cloning which takes longer and is less efficient
what can PCR be used for
- Generation of hybridization probes
- DNA cloning
- DNA sequencing
- Isolation of DNA for recombinant technologies
- Rapid screening of colonies
- Genetic fingerprinting
- Paternity testing and evolutionary relationships between organisms
what is a primer (or oligonucleotide)
strand of nucleic acid that functions as the starting point for DNA synthesis; in vivo the primer is made of RNA
what is DNA polymerase
enzyme that is involved in polymerization of deoxyribonucleotides into DNA. The new synthesised strand is complementary to the template strand
what does PCR reaction require
- ds DNA template
- A heat-resistant DNA polymerase
- Four nucleotides: dATP, dTTP, dCTP and dGTP
- Two short single-stranded DNA molecules serving as primers and complementary to each one of the strands of DNA
- Magnesium ions (act as cofactors for DNA polymerase)
- Buffer containing salt (DNA is acidic)
what are the steps in PCR
- Heat (94-99°C) to denature DNA strands
- Cool (50-65°C) to anneal primers to template
- Warm (68-72°C) to activate Taq polymerase, which extends primers and replicates DNA
- Repeat multiple cycles
how does DNA template denature
heat cause DNA strands to separate
occurs at 94 degrees C
what is annealing
pairing of DNA or RNA by hydrogen bonds to a complementary sequence to form a double-stranded polynucleotide
what are occurs during annealing
- Primers bind to the template sequence at 60 degrees
- Taq polymerase binds to double-stranded substrate
what happens at 72 degrees
Taq Polymerase extends the primer
DNA is replicated
when is the exact target product made
in third cycle of PCR
what happens in cycle 1 of PCR
yields two molecules
what happens in cycle 2 of PCR
yields four molecules
what happens in cycle 3 ofPCR
yields eight molecules: 2molecules match the target sequence
what is Taq polymerase
Heat resistant – enzyme from Thermus aquaticus (living in hot springs) and withstands heat ≽ 100oC
what is the error rate of replication fidelity of taq polymerase
error rate 1/10000 nucleotides –> produces 16% mutations in DNA segment of 1kb
what is the replication of fidelity of taq polymerase
fast –> can amplify 1kb strand of DNA in ~ 30 secs at 72 degrees C
what does the taq polymerase lack
lacks 3’ to 5’ exonuclease proofreading mechanism
what is pfu polymerase
Heat-resistant, hyperthermophilic, has been isolated from the archaeon Pyrococcus furiosus and withstands heat > 100oC
what is the error rate of replication fidelity of pfu polymerase
proofreading activity (error rate of 1/1.3 million bp and yields only 2.6% mutations in a DNA segment of 1kb) because it has a 3’ to 5’ exonuclease mechanism which corrects mis incorporated nucleotides –> lower errors –> high
what is a disadvantage of pfu polymerase
Slower –> requires 1–2 mins to amplify
what does pfu produce and what what is required
1kb of DNA at 72° C fidelity
what can provide best characteristics for PCR
Mixing both Taq polymerase + Pfu polymerase it is possible to obtain both the fidelity of Pfu and the speed of Taq
how many base pairs should primers be
15 bp long
how much of the primers is made up of GC
40-60%
what temperature do primers anneal at
50-60 degrees C
what temperature should forward and reverse primers anneal
approx same temperature
what is the primer sequence
they should be unique(if a primer matches multiple sequences multiple products will result)
should not have self-annealing regions in their sequences (or will anneal to each other, so will not anneal to DNA want to amplify)
should not have self-annealing regions in their sequences (hairpin and foldback loops can be generated)
what will happen is the pairs of primers anneal
they will form primer dimers
what are the PCR types
DNA PCR RNA PCR Multiplex PCR In situ PCR Real time PCR
what is DNA PCR
amplification from DNA
what is RNA PCR
amplification from RNA via Reverse Transcriptase reaction followed by DNA PCR
what is multiplex PCR
different primer combinations simultaneously to detect & differentiate agents
what is in situ PCR
localization of gene/ transcript
what is real time PCR
real-time detection using various dye chemistries
what is the difference in conventional and real time PCR detection
C - end point detection
RT - exponential point detection
what is the difference in conventional and real time PCR for precision
C - poor precision
RT - high precision
what is the difference in conventional and real time PCR sensitivity
C - low
RT - high
what is the difference in conventional and real time PCR automation
C - non automated
RT - highly automated
what is the difference in conventional and real time PCR quanification
C - difficult to quantify
RT - quantification
what is the difference in conventional and real time PCR discrimination
C - Discrimination based on size
RT - Discrimination based on labeled probes, Tm
what is the difference in conventional and real time PCR safety
C - carcinogenic
RT - safe
what is the difference in conventional and real time PCR
C - post-PCR processing
RT - closed format, results and analysis obtained in real-time
where is real time detection
in beginning of cycle is exponential
where is the conventional PCR detection
in plateau area
what is real time PCR able to detect
two-fold change (i.e. 10 Vs. 20 copies)
what are the realtime PCR chemistries
DNA binding dyes
Probe-based
what are the DNA binding dyes
SYBR green
Bebo
what are the probe based
TaqMan
Molecular beacon
what is SYBR green dye used for
identify real time
what does SYBR do
will bind to any double-stranded DNA molecule, while the 5’ Nuclease assay is specific to a pre-determined target
attaches any dsDNA including primer-dimers
what increases the SYBR green sye signal
more ds amplicons
what happens in SYBR green and polymerization
- DNA is denatured, the SYBR Green dye floats free
- Extension phase begins as primers anneal
- SYBR Green dye binds to the ds products / amplicons and fluoresces
what is the SYBR green real time PCR
critical parameters - threshold value
melting temperature
what is the threshold value
- threshold line is the level of detection (set in the exponential phase of the amplification)
- cycle threshold, Ct
what is the melting temperature (Tm)
- Temperature at which 50% DNA is denatured
- Only required for SYBR Green assay to check specificity
what is the cycle threshold
directly proportional to amount of starting template
how can the concentration of unknown sample be found
comparing Ct to a standard curve generated by dilutions of known amounts of a product
what happens there is a high starting amount of nucleic acid
Higher the starting amount of the nucleic acid target, the sooner a significant increase in fluorescence is observed
what is the melting curve analysis influenced by
Tm of a specific amplification is influenced by:
- length of the Amplicon
- Nucleotide sequences of the Amplicon (GC/AT ratio)
- PCR conditions
what is a TaqMan probe
hydrolysis probes designed to increase the specificity of real-time PCR assays
what happens in TaqMan probe use to the dye
Reporter dye’s emission is suppressed by the Quencher dye
what happens when the enzyme reaches the TaqMan probe
the enzyme 5’ exonuclease activity begins
what happens after exonuclease activity begins in TaqMan probe use
Reporter starts to fluoresce as it is separated from the Quencher
