Term
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Definition
| Amplification of target DNA sequence in vitro. This is preferable to growing many cell in a culture and waiting until there are enough to visualize. |
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Term
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Definition
- Target dsDNA template
- Deoxynucleotide triphosphates (dNTP) - dATP, dGTP, dCTP, dTTP
- DNA Polymerase
- Primers
- Buffer
- MgCl |
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Term
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Definition
Denaturation - 90-96 C for 20-60s. dsDNA template is denatured to ssDNA. Initial denaturation for long DNA fragments is longer.
Annealing - 50-70 C for 20-90s. Important for specificity. Primers hybridize to ssDNA template. This takes primer Tm into account for optimal conditions.
Extension - 68-75 C for 10-60s. DNA polymerase adds on to primers, synthesizing DNA.
At the end of every cycle of these steps, target DNA is doubled. |
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Term
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Definition
20-30 bp ssDNA sequences flanking to region of interest. Determine the specificity of PPCR, kind of like probes.
Forward primer attaches to the template DNA 5' of the target sequence.
Reverse primer attaches to the template DNA 3' of the target sequence.
Primer placement determines the length of the PCR product. |
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Term
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Definition
%GC and primer length affect what conditions are optimal for hybridization. Tm is calculated by
(4 C x +GC pairs) + (2 C x #AT pairs)
Tm is a guideline for annealing temperature, and can be adjusted by changing primer length and %GC.
Reverse and forward primers should have the same Tm. |
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Term
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Definition
| Primers can bind to the wrong sequence to produce non-target DNA sequences that contain the complimentary primer sequences. This incorrect fragment can be amplified in subsequent cycles and compete with target DNA for primer binding. They can also interfere with sequencing and data analysis. |
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Term
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Definition
| Secondary structures can interfere with PCR, as can primer pairs with homologies. If the homologies are at the 3' end of the primers, reverse and forward primers can bind and produce DNA fragments twice the length of the primers. This is an efficient competitor with the target sequence for amplification. |
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Term
| Primer Binding Requirements |
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Definition
| The whole primer sequence doesn't have to be strictly complimentary to the target sequence, but the 3' end must be, so that extension of the primer by DNA polymerase is possible. The final 3' nucleotide must be H-bonded to the template for a phosphodiester bond to form. |
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Term
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Definition
| Primers that are not complimentary to the target strand on their 5' end. This "tail" will then be added to all subsequent products. Restriction sites, promoters, or binding sites can be added. One or both of the ends of the PCR product can be added on to. |
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Term
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Definition
DNA should be intact, without nicks or breaks, and free of proteins. High GC content and secondary structure can interfere.
Most cells only have one or two copies of any gene. 100 ng - 1 ug of DNA is usually required for PCR. |
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Term
| dNTP Requirements for PCR |
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Definition
The four dNTPs are added in equimolar amounts. Enough of each is needed to support ever increasing copies of the template. 021-025 mM concentration is usually enough. Storage concentration should be higher, 10-100 mM.
Altered nucleotides like denza dGTP (destabilizes secondary structures of GC binding) can be added. |
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Term
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Definition
DNA Polyemrase lacking N-terminal 289 AA and 3'-5' exonuclease activity.
Has a long half-life at high temperatures.
Works in a wide range of MgCl2 concentrations.
Good for allele-specific PCR or regions with high GC Content. |
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Term
| Taq Polymerase Disruption |
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Definition
| Taq works at very high temperatures, but it can lose its function if agitated too much. Vigorous shaking or mixing can cause shearing and altering of secondary and tertiary structures. This denaturation is irreversible. Most polymerases are multi-subunit enzymes, so if they lose their shape they lose their function. |
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Term
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Definition
Provides correct conditions for optimal enzyme activity and target amplification. Sources of noncovalent cations (KCl 20-100 mM, ammonium sulfate 15-30 mM) are important components. Salts will affect annealing and denaturation of DNA. High salt concentration makes long DNA fragments denature slowly, allowing short fragments to be preferentially amplified.
Tris buffer maintains a pH of 8-9.5. |
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Term
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Definition
MgCl2 affects primer annealing and enzyme activity. One Mg ion is needed for each dNTP.
Optimal MgCl2 concentration is 1-4 mM. Too high and nonspecific dNTPs will be incorporated, too low and enzyme activity will decrease.
EDTA and other chelating agents reduce the amount of MgCl2 available to the polymerases, reducing amplification efficiency. |
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Term
| Bovine Serum Albumin in PCR |
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Definition
| At 10-100 ug/mL will stabilize enzymes and bind inhibitors. |
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Term
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Definition
| At 0.01 mM will create reducing conditions that may be conducive to enzyme activity. |
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Term
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Definition
| 1-10% will denature dsDNA secondary structures and lower the temperature required to denature DNA. |
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Term
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Definition
| Triton X-100, glycerol, dimethyl sulfoxide. At 1-10% will reduce DNA secondary structure and allow polymerase to work, increasing enzyme stability. |
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Term
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Definition
Positive Control - shows that buffers, primers, enzymes all performed optimally.
Amplification Control - Second set of primers and non-target DNA. Shows that the reaction is working and helps distinguish false from true negatives.
Negative Control - No DNA, shows that there's no DNA contamination.
Negative Template Control - DNA without the primer target sequences. Shows that primers are annealing accurately. |
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Term
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Definition
Can be used to decontaminate pre-PCR areas. Makes single and double-stranded breaks in DNA, preventing replication. It can work in conjunction with P. soralens, which intercalates with dsDNA and binds to uracils, thymines, and cytidines in the presence of UV light, preventing denaturation and amplification.
UV light can damage people and plastics. If it is far away from a surface it won't do much good. |
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Term
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Definition
dUTP can be added during PCR instead of dTTP. The amplicons will have U instead of T, which won't affect the final product in any negative way.
Uracil-N-glycosylase (UNG) can be added for a short incubation (2-15 min at 50C) at the start of PCR. It will degrade U in nucleic acids, getting rid of amplicons from prior reactions.
This doesn't work for nested PCR, but is good for RT-PCR, where contaminants are indistinguishable from positives. |
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Term
| Mispriming and Amplification Conditions |
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Definition
| Good primer design and optimal annealing conditions prevent much mispriming. Mispriming can occur before PCR, when the reaction mix is being assembled. Taq at room temperature still has some functionality, so when its added to the DNA/Primer mix at low temperatures, stringency conditions are low and primers may attach to the wrong sequences and produce misprimed products that are then extended in PCR. |
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Term
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Definition
Three methods used to prevent mispriming
1. Reactions are prepped on ice and put in the thermocycler already at a hot temperature.
2. Wax beads are added to the mix before the template DNA and DNA polymerase are added. The tubes are heated to 100 C so that the wax melts. When the mix cools to room temp, the wax cools and forms a layer at the top of the tube. DNA template and polymerase are added on top of the wax, which will then be melted in the first step of PCR. After PCR the wax must be punctured.
3. Polymerases are sequestered with antibodies or some other agent that will deactivate them. The polymerase won't work until the denaturation heat is reached. |
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Term
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Definition
| The initial annealing steps of PCR are done at a temperature much higher than the optimal primer binding temperature (high stringency). The temperature drops 1 C every cycle until the correct temperature is reached. This favors correct primer binding over mispriming. |
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Term
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Definition
| Amplicons can be run via gel electrophoresis and the amplicon bands cut out. The gel can be dissolved by beta-agarose enzymes or by mixing with iodine after centrifuging. The result will be free of primer dimers. |
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Term
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Definition
| DNA is applied to columns where it binds and the other PCR components are washed away. This allows for the removal of buffer or nucleotides that might interfere with post-PCR reactions. Won't get rid of all residual primers or misprimed products. |
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Term
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Definition
SAP and ExoI are added post PCR. SAP dephosphorylases nucleotides and ExoI degrades primers. This happens at 37 C for 15 min, followed by a 80 C 15 min incubation to denature the proteins.
Doesn't remove PCR bugger or MgCl2. |
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Term
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Definition
| Multiple Primers are included so that multiple amplicons are produced by each sample. This can be used to identify multiple viruses in one sample or to identify both an organism and what drug resistance it may have. |
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Term
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Definition
| Hard to get right. Multiple primers might have different optimal annealing temperatures or might interfere with the binding of other primers. Multiplexed controls can be added to tests determining presence or absence of product. Primers and controls should be chosen so that they don't interfere with each other. |
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Term
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Definition
The 3' ends of primers must be strictly complimentary to the DNA sequence. If a single base at the 3' end is changed, it can be used to detect the presence or absence of specific mutations.
The high sensitivity of the test means that it may pick up mutations that are not present in high enough numbers to be clinically significant. |
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Term
| Reverse Transcriptase PCR |
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Definition
RT-PCR. ssRNA is not a good template for amplification, but it can be converted to dsDNA by reverse transcriptase (RT). RT copies the RNA, making a DNA:RNA hybrid, and then uses a hairpin at the end of the strand to double back and replace the RNA with DNA. This produces complimentary (copy) DNA (cDNA).
Used to analyze RNA expression, rRNA detection, analysis of genes with long introns, (cDNA has no introns), and to detect rRNA genomes of microorganisms. cDNA make particularly good sequencing templates, since there are no introns. |
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Term
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Definition
Specific oligo dt primers or hexamers are often used to prime synthesis of specific DNA strands, but the cDNA yield is low.
Non-specific oligo dt primers are 18 base long polyT sequences that prime DNA synthesis from mRNA molecules with polyA tails. This gives a high but unspecific yield.
Random 6-10 b rimers (hexamers/decamers) can be used to achieve the highest yield. This generates cDNA from all the RNA in the sample, so is not specific. |
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Term
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Definition
| RT PCR can be done in two steps - RT and then PCR - but the addition of Tth DNA polymerase with RT activity and hot-start DNA polymerase can simplify it to one step. And initial incubation of 30-60 min at 45-50 C allows the RT to synthesize cDNA, and then the first step of PCR will denature the RT. |
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Term
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Definition
Increases sensitivity and specificity of PCR. Particularly useful for clinical samples with low levels of target DNA and high levels of interfering sequences.
PCR is run twice, with a different primer pair each time. The second set of primers is complimentary to the sequence slightly inside of the sequence targeted by the initial primers. This increases specificity.
One of the second primers can match one of the first primers (semi-nested PCR). |
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Term
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Definition
| 5' tails can be added to the first round of primers that compliment the sequence of second round primers. Useful for multiplex reactions where all the initial primers may differ in bonding efficiency. All products will be amplified by the second set of primers, increasing sensitivity but not specificity. |
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Term
| PCR Product Quantification via Competitors |
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Definition
Competitors are test sequences of a known amount that are amplified in the same reaction as the samples. The ending ratio of target DNA:competitor and the known initial concentration of the competitor can be used to calculate the initial DNA concentration. Primer incompatibilities and inconsistent results plague this approach.
Multiple dilutions of the competitor can be added to the reaction to assess target DNA amplification compared to the internal control. This doesn't work if there's a 10-fold difference between the two. |
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Term
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Definition
| Used for most PCR methods. The analysis of PCR products happens at the plateau phase, when reaction components are exhausted and all amplification products have reached a similar concentration, even if they started at different concentrations. |
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Term
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Definition
| qPCR. Dyes (such as EtBr or SYBR Green) are added to the PCR reaction and fluoresce as copy numbers increase. The amount of cycles it takes for the level of fluorescence to cross a threshold is inversely proportional to the number of copies at the start of PCR. The threshold cycle (Ct) can be used to find the number of starting copies. |
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Term
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Definition
Nonspecific dyes such as EtBr and SYBR Green can be used, but they will bind to all dsDNA including misprimed products and primer dimers. They can only be used in very clean reactions.
Probes can be used to generate fluorescence from specific sequences only. |
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Term
| SSOP Reverse Configuration |
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Definition
| Primers in PCR are attached to biotin or digoxygenin at the 5' end, so amplicons contain these molecules. Multiple alleles are attached to the membrane, so the patient sample is applied to see where the amplicons bind. |
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Term
| Whole Genome Amplification |
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Definition
WGA. Uses non-specific primesr to start replication at various points in the template to survey all genes/transcripts present. Can be used to type organisms or on samples with low concentrations of DNA.
Can be used for comparative genome arrays, detection of SNPs, organism typing, and ancient DNA samples. |
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Term
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Definition
| Single-stranded breaks or nicks are introduced in the genomic material. Random hexameric primers are added to start amplification. Amplification can laso start at the 3' end of nicks. DNA ahead of the nick will need to be degraded as extension proceeds (multiple displacement amplification). Phi29 DNA polymerase is used because it is highly progressive (stays on the template for 1,000s of bases even as it displaces nucleotides). |
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Term
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Definition
| WGA uses degenerative primers to amplify all regions of the template. Primers have a sequence that occurs frequently throughout the genome, with some nucleotides that vary. |
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Term
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Definition
Used to simultaneously amplify many unique sequences in one tube. Template DNA is fragmented and then ligated to adaptors that match universal primers. All PCR components are added to an oil surfactant mix. Stirring fragments the mix into many droplets, each of which contains a single DNA fragment and PCR components. The droplet serves as a mini-reaction chamber, amplifying that fragment. When amplification is done the emulsion is broken and the fragments are pooled.
Used for arrays, haplotyping, and rare mutation detection. |
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Term
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Definition
| Primers are immobilized to beads and added to DNA fragments with adaptors. These and PCR components are added to a water-in-oil emulsion mix, creating aqueous droplets in the oil that contain one fragment and one bead. PCR products are washed away after emulsion, leaving a ssDNA that can be used for Next Gen sequencing. |
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Term
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Definition
| Bridge PCR. Primers are attached to a flowcell. Template DNA is denatured and added. Primers are extended at 60 C, and formamide is added to denature dsDNA. The template and reaction components are washed away. Annealing conditions are reestablished so the template will attach to the reverse primer, which will in turn extend and form a bridge. This is repeated 35 times for 1,000 copies. dsDNA is then denatured, leaving many clones of the complimentary sequence. Primers contain a cleavable site for removal. |
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Term
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Definition
The number of target molecules isn't amplified. Instead, a synthetic probe binds to the target sequence and is amplified.
Ligase Chain Reaction
Strand Displacement Amplification
QB Replicase |
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Term
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Definition
LCR. Primers have biotin attached to one primer and a signal producing molecule on the other. Primers bind to the target sequence directly adjacent to each other. DNA Ligase joins the two together, and the ligated primers can now serve as a template.
A Thermocycler is required for the hot denaturation step and the cool annealing/ligating step. A one pair mismatch between the primer and the template will prevent binding, allowing for detection of point mutations. |
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Term
| Strand Displacement Amplification Step One |
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Definition
| An isothermal reaction. Target DNA is denatured at 95 C. Two primers anneal to the target sequence, the outer bumper probe and the inner probe with a restriction site tail on the 5' end. Exonuclease deficient DNA polymerase extends both primers. The stran from the bumper will displace the strand started by the inner probe. Modified nucleotides are included in this synthesis. The displaced strand with the restriction site is amplified using a second set of primers to make a structure that contains a normal restriction site and a restriction site with modified nucleotides. This ds species is the target for amplification. |
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Term
| Strand Displacement Amplification Stage Two |
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Definition
| Restriction enzyme HincIII cutes the normal restriction site on the ds species but can't cut the restriction site with modified nucleotides. DNA polymerase will sense the nick and come extend it, displacing the original strand. This makes a new single-stranded probe and regenerates a dos species with the restriction site on one strand. The ds species don't need to be denatured, so it all takes place at 52 C |
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Term
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Definition
| First used to detect M. Tuberculosis. Fluorescent polarization can be used to detech M. tuberculosis and C. trachomatis. The use of fluorogenic probes allows for the quantification of the amount of target. Also used for N. gonorrhoaea. |
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Term
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Definition
An RNA-Dependent RNA polymerase. RNA or denatured DNA is mixed with reporter probes containing the QB promoter sequence and the target sequence. These probes and a polyC probe attach to the template and the polyC probe attaches the complex to a polyG magnetic bead. Unbound probes are washed away and then the complexes are denatured from the bead. A second wash can be done with a polyA probe and a polyT bead. Once the complex is removed from the beads, QB can be added to replicate the probe molecules by 10^6 - 10^9 in 15 minutes.
This produces so many probes that colorimetric detection can be used. |
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Term
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Definition
Targets and probes aren't amplified. Lots of signal molecules are attached to the template available in the clinical sample, reliably quantifying the amount of template.
Branched DNA Amplification
Hybrid Capture Assays
Cleavage-Based Amplification
Cycling Probe |
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Term
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Definition
| Probe-based qPCR. A probe complimentary to a sequence in the target region is added to the reaction mix in addition to the primers. The 3' end is modified so that DNA polymerase can't extend it. The 3' end is attached to a quencher, a nonfluorescent dye that pulls fluorescence away from the dye at the 5' end. When taq polymerase reaches the probe it will degrade the probe with its 5'-3' exonuclease activity. When the quencher has been separated from the probe, the probe will generate fluorescence. |
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Term
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Definition
| Probe-based qPCR. The probe is a 25 b sequence complimentary to the target. The 5' end has a fluorophor and the 3' end has a quencher. Each end has an inverted repeat. When the probe is free, the repeats bind, forming a hairpin structure where the quencher can affect the dye. During the annealing step the probe binds to the target DNA, separating the probe and the quencher so fluorescence is generated. During extension the primers will displace the probe and it will return to a hairpin state. |
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Term
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Definition
| Variation of molecular beacons used for qPCR. The target-specific primer is attached at the 5' end to a hairpin structure with a dye and a quencher at either end. When the primer is unbound and intact, the quencher mutes the fluorophor. When the primer hybridizes to the target sequence and is extended, the hairpin structure is straightened out, removing the quencher from the dye's presence. This is a fast method. The fluorophor is incorporated into the amplicon. |
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Term
| Fluorescence Resonance Energy Transfer |
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Definition
FRET. A probe-based qPCR. Two probes are used, one with a 3' fluorophor and one with a 5' donor. Common pairs are fluorescein-rhodamine, fluorescein-(2 aminopurine), and fluorescein-Cy5.
When the two probes bind to the target sequence and the donor and dye are in proximity, the donor transfers energy to the fluorophor and fluorescence is generated.
Alterations in the probe can be used to detect mutations or organisms. |
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Term
| Transcription - Based Amplification System |
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Definition
TAS. Target RNA has DNA copied from it. The cDNA is then transcribed to produce numerous RNA copies. RNA is the target and the product.
Useful because:
1. Isothermal, no thermocyling
2. RNA viruses can be detected
3. Organisms with little DNA but many RNA products are detected |
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Term
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Definition
A primer with an RNA polymerase binding site on one end binds to the RNA template, enabling RT to make a DNA copy. The DNA:RNA hybrid is denatured with heat and a different primer binds to the DNA and is extended by RT. RNA polymerase T7 transcribes the DNA, making many RNA copies which then serve as more templates for cDNA synthesis.
The heating step that degrades DNA:RNA hybrids also denatures proteins, so new enzymes must be added at each cycle. |
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Term
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Definition
Transcription-Mediated Amplification/Nucleic Acid Sequence-Based Amplification, or self-sustaining sequence replication (3SR).
An improved TAS procedure where the denaturation step is eliminated with the addition of RNAse H, which degrades RNA in DNA:RNA hybrids. A RT from avian myeloblastosis virus (AMV) has RNAse H capabilities, so only AMV RT and T7 RNA polymerase are needed.
NASBA can be used on DNA targets. |
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Term
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Definition
Randomly Amplified Polymorphic DNA (RAPD).
Low stringency conditions and short, random primers are added to amplify arbitrary genomic sequences. No specific gene is targeted. Many short fragments may be amplified, depending on how often the primer sequences appear in the genome. Can be used to type organisms and find similar ones.
Low stringency means that results may not be repeatable on any two samples. |
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Term
| Branched DNA Amplification |
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Definition
| bDNA. Target DNA is denatured. Capture probes with sequences complimentary to the target bin to it and attach to the plate well. Extender/preamplifier probes that compliment a different part of the target bind. These probes also contain a sequence that is complimentary to amplifier probes. Amplifiers have hyvridization sites for 8-14 brances, which bind to substrate molecules from AP. |
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Term
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Definition
| Target nucleic acid fragments bind to capture probes and are immobilized. Extender probes bind to the target and also bind to pre-amplifier sequences, which can bind to 14-15 amplifiers each. Amplifiers can bind to multiple AP-labelled nucleotides. Dioxetane is added for AP to act on. A luminometer can read fluorescence down to 50 target mol/mL. |
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Term
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Definition
-Carryover contamination is less likely to results a false positive
- Different capture/extender probes can be added to detect variations of sequences, such as disease isolates.
- Multiple probes must bind to the target, ensuring high specificity.
Used for HepB, Hep C, HIV |
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Term
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Definition
Denatured DNA is added to an RNA probe. Antibodies on the well wall recognize and bind to the DNA:RNA hybrid, but not dsDNA or ssRNA. AP-conjugated antibodies attach to the hybrid. AP substrate is added and chemiluminescence is measured.
A signal amplification assay.
Sensitive down to 1,00 copie of HPV. Also used for HepB and CMV. |
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Term
| Cleavage-Based Amplification |
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Definition
| Used to detect DNA polymorphisms, HCV and HPV genotyping. Target nucleic acid is hybridized to an invader probe and a signal probe. Cleavase recognizes the two probe overlap and cleaves the signal probe, which becomes a second invader probe. FRET probes are added that match the second invader probe. Intact FRET probes have a fluorophor molecule at the 5' end, located near a quencher. When the invader probe attaches to the FRET probe, it makes an overlap cut by cleavase. This frees the reporter from the quencher, so signal is produced. |
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Term
| Cycling Probe Amplification |
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Definition
ssSynthetic probes of DNA-RNA-DNA with a reporter dye at one and and a quencher at the other are added and hybridize to the target nucleic acid. RNase H degrades the RNA portion. The hybridization temp of the two DNA probes is lowered, so they de-attach. This frees the fluorophor from the quencher. The target can be bound again. An isothermal reaction. Fluorescence is measured to indicate the presence of target. If the target isn't present, the RNA:DNA chimeras can be detected.
Used to detect antimicrobial genes. |
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