PCR Flashcards
PCR
Polymerase chain reaction.
Target Amplification
Making many copies of a specific DNA sequence.
Target (PCR)
A short region of DNA
Amplicons
Copies of DNA
Amplification Program
A specified number of cycles that are divided into steps during which the samples are help at a particular temperature for an amount of time.
Cycles (PCR)
One series of denaturation, annealing, and extension.
Annealing
Second step in PCR where two oligonucleotide primers prime the synthesis of DNA anneal to complementary sequences on the template.
Mispriming
Aberrant initiation of DNA synthesis from a primer hybridized to template sequences different from the intended target.
Primer Dimers
PCR products that are approximately double the size of the primers.
Taq Polymerase
Thermostable enzyme from the thermophillic bacterium Thermus aquaticus.
Tth Polymerase
Enzyme from Thermus thermophilis. Also has reverse transcription activity.
Stoffel Fragment
Modified version of the polymerase enzyme.
Real-time PCR
PCR performed in an instrument that can measure the formation of amplicons in real time. AKA Quantitative PCR. Will inform if the the target sequence is found.
Negative Template Control
A negative control with DNA that lacks the target sequence to ensure that the primers are not annealing to nontarget sequences of DNA.
False Negative
Amplification failure.
Psoralen
A substance from plants that covalently attaches to DNA (it attaches to the thymidines, uracils, and cytidines in the DNA chain) under UV light. Helps with the prevention of denaturation and amplification of treated DNA.
Hot-start PCR
Used to help prevent mispriming.
Three methods:
1. Mix reactions on ice and add to a preheated thermocycler.
2. Wax was used in older thermocyclers as a barrier to prevent evaporation.
3. Use of sequestered enzymes. These will not activate unless heated by the first denaturation step.
Touchdown PCR
A modification of the PCR program. Annealing temperatures begin with a higher than optimal temperature for target primer-binding. The annealing temperature is then lowered by 1 degree every cycle or every other cycle until the optimal temperature is reached.
Hybrid in PCR
DNA/RNA hybrid
Multiplex PCR
More than one primer is being used in PCR.
Nested PCR
PCR with increased sensitivity and specificity. 2 pairs of primers are used to amplify a single target in 2 separate PCR runs. The second priming pair binds just inside the first one.
Semi-nested PCR
One of the second round primers is the same as the first one.
Endpoint Analysis
Analysis or observation of a PCR product after the amplification program is complete.
Ct
Threshold cycle. The cycle at which sample fluorescence crosses the threshold.
SYBR Green
Dye specific to double stranded DNA. Bind and fluoresce specifically in the double-stranded DNA product of the PCR. Advantages are its specificity and robust fluorescence comparable to EtBr and its reduced toxicity.
EtBr
Dye originally used in PCR.
TaqMan
A probe detection system used in real-time PCR. This method exploits the 5’ to 3’ exonuclease activity of Taq polymerase to generate a signal.
Quencher
A molecule that can take fluorescent energy from a reporter dye. Used in the taqman procedure.
Molecular Beacons
A probe-based detection system that measures the accumulation of product at the annealing step in the PCR cycle.
Scorpions
A variation of molecular beacons that produces a PCR product that is covalently labeled with the fluorophore that can further be analyzed by capillary electrophoresis.
FRET
Fluorescent Resonance Energy Transfer. A PCR system that utilizes two probes that bind to adjacent targets. Relies on the transfer of excitement energy from a donor florophore to an acceptor florophore.
Transcription Based Amplification Systems
TAS. RNA is the target instead of DNA. A DNA copy is synthesized from the RNA and then transcription of the DNA produces millions of copies of RNA.
Genomic Amplification Methods
Amplifies all regions of the input DNA.
Comparative Genomic Arrays
Detection of single-nucleotide polymorphisms, and analysis of minimal starting material, such as DNA in plasma, single-cell analysis, and ancient DNA samples.
Emulsion PCR
Simultaneously amplifies thousands of specific templates in a single reaction, producing a library.
Surface Amplification
Bridge PCR. An isothermal, genomic PCR method used in high-throughput sequencing technologies. Forward and reverse primers are immobilized on a solid support in a flow cell.
Isothermal
Involving or consisting of a constant temperature.
Polony
A localized clone.
Arbitrarily Primed PCR
Randomly Amplified Polymorphic DNA. Random Amplification of Polymorphic DNA (RAPD). Short primers with random sequences are used to amplify arbitrary regions in the genomic DNA under low-stringency conditions. PCR products are generated without knowing the sequence of the target or targeting a specific gene.
Probe Amplification
The number of target nucleic acid sequences in a sample is not changed. Synthetic probes that are specific to the target sequences bind to the target where the probes themselves are amplified.
Ligase Chain Reaction
LCR. A type of probe amplification. A method for amplifying synthetic primers or probes complementary to target nucleic acid. The entire target sequence has to be known.
Strand Displacement Amplification
SDA. An isothermal amplification process, where after the initial denaturation step, the process proceeds at one temperature. The major amplification products are the probes.
QB Replicase
A method for amplifying probes that that have specificity for a target sequence. The method is named from the major enzyme that is used to amplify probe sequences.
Restriction Map
A diagram showing the linear placement of restriction enzyme recognition sites in DNA.
Restriction Fragment Length Polymorphisms
RFLP. The resulting differences in the size or number of restriction fragments.
Single-nucleotide Polymorphism
SNP. Substitution of a single nucleotide.
Signal Amplification
Large amounts of signal are bound to the target sequences that are present in the sample. No change in the amount of target or probe sequences. Amplification of the probe signal rather than the target.
Branched DNA Amplification
bDNA. A series of short oligomer probes is used to capture a single target nucleic acid molecule.
Hybrid Capture Assays
Target DNA released from cells is bound to single-stranded RNA probes creating a DNA-RNA hybrid and is recognized by antibodies.
Cleavage-Based Amplification
Detects target nucleic acids by using a series of overlapping probes that bind to the target DNA. A signal amplification system.
Cycling Probe
A signal amplification system. Target sequences are detected using a synthetic probe consisting of sequences of DNA and RNA arranged in a RNA-DNA sandwich sequence carrying a reporter dye at one end and a quencher dye at the other. RNase H digests the RNA probe releasing the reporter dye from the quencher, creating fluorescence.
mecA
methicillin resistance
