Competitive PCR: is a a type of quantitative PCR. Exogenous target is added to create a competition between templates for amplification. The exogenous target is designed to amplify a smaller fragment with the same primer set so that products can be visualized easily by agarose gel electrophoresis and quantitated. The ratio of products obtained from exogenous and sample targets reflects the initial ratio of target to competitor.
DNA Fingerprinting Analysis: Amplification of regions of with sequence specific primers produces a banding pattern that can serve as a “fingerprint” to uniquely identify individuals. PCR-based restriction fragment length polymorphism (PCR-RFLP): The scheme of includes amplification of DNA containing the possible mutation or a known polymorphism using flanking primers, followed by restriction digestion of the PCR product. Banding patterns of the amplified fragments will show the mutation. Thus this PCR technique can be used to detect genetic mutations.
Long PCR: PCR reactions that are optimized for longer templates. This can be done by combining different DNA polymerases to increase processivity and fidelity of the reaction as well as increased extension times and the addition of cosolvents.
Multiplex PCR: PCR reactions where multiple sets of primers are used to amplify more than one target DNA. Thus, more than one unique target DNA sequence in a specimen can be amplified at the same time. Primers used in multiplex reactions must be carefully designed to have similar annealing temperatures, which often requires extensive empirical testing. Quantitative competitive PCR, a variation of multiplex PCR, can be used to quantify the amount of target DNA or RNA in a specimen.
Nested PCR: Nested PCR, designed mainly to increase sensitivity (detect small quantities of taarget), uses two sets of amplification primers. One set of primers is used for the first round of amplificaiton, which consists of 15 to 30 cycles. The amplification products of the first reaction are then subjected to a second round of amplification with another set of primers that are specific for an internal sequence that was amplified by the first pirmer pair. The major disadvantage of the nested amplificaiton protocol is the high probabiltiy of contamination during transfer of the first round amplificaiton products to a second reaction tube. This can be avoided either by physically separating the two amplifcaiton mixtures with a lawyer of was or iol, or by designing the primer sets to utilize substantially different annelaing temperatures.
Degenerate PCR: Instead of using specific PCR primers with a given sequence, you use mixed PCR primers with degenerate PCR. In other words, you insert “wobbles” in the PCR primer. The degeneracy of the primer is introduced during DNA synthesis.
Degenerate PCR has proven useful to find related genes in families.
RT-PCR: combines reverse transcription and PCR. This brings the benefits of PCR to analysis of RNA. In this process, RNA tragets are first converted to complementary DNA (cDNA) by RT, and then amplified by PCR. RT-PCR can be used for amplification based cDNA library construction.
RNA Purification Kits: Gentra Systems
Differential Display: is a gene expression analysis method whereby mRNA from each sample is converted to cDNA which is then PCR-amplified using a combination of random primers (8-13mers) and anchored oligo-dT primers. The products are run on a gel. Each mRNA is represented as a signal band and differentially expressed bands are excised, cloned, and sequenced to reveal identity.
Ligase Chain Reaction (LCR): is a probe amplification technique. Successful ligation relies on the contigous positioning and correct base pairing of the 3′ and 5′ ends of oligonucleotide probes on a target DNA molecule. In the process, oligonucleotide probes are annealed to template molecules in a head to tail fashion, with the 3′ end of one probe abutting the 5′ end of the second. DNA ligase then joins the adjacent 3′ and 5′ ends to form a duplciate of one strand of the target. A econd primer set, complementary to the first, then uses this duplciated strand (as well as the original target) as a template for ligation. Repeating the process results in a logarithmic accumulation of ligation products, which can be detected by means of the functional groups atached to the oligonucleotides.
Multiplex qPCR: refers to simultaneous quantification of multiple templates int he same reaction and depends on the ability to measure a different fluorescent signal for each nucleic acid target, often by using multiple TaqMan probes labeled with different fluorophores. One important applicaiton of multiplex RT-qPCR is the concurrent amplificaiton of a target gene and a housekeeping gene in the same reaction.
RACE PCR: amplifies 5′ and 3′ UTRs. For 3′ UTRs, one can use an oligo (dT) adapter linked primer to reverse transcript mRNA.
Real Time PCR: incorporates the ability to directly measure and quantify the PCR reaction products while the amplification is taking place. RT-rt-PCR has several advantages over conventional competitive RT-PCR. 1) it is up to 100 times more senstivie then conventional PCR; 2) it has a higher specifictiy because of three sequence specific oligonucleotides involved in the amplification process; 3) quantification is always performed in the log phase of PCR; 4) the linear range of quantification is wider; 5) there is almost no risk of cross-contamination with amplicons; 6) the detection of faulty amplifications is easy by software-based on-line verification of the overall quality of the PCR run.
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intercalating dyes are typically used. SYBR Green is the dye of choice. It fluoresces 200 times more brightly when bound to dsDNA. Since SYBR Green will intercalate into any DNA in your reaction (ie., primer Dimers, contaminating DNA), you should do a melt curve analysis.
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Using Hybridization Probes using TaqMan and Molecular Beacons. The TaqMan 5NA (Perkin Elmer/Applied Biosystems) utilizes an oligonucleotide probe, whose sequence is selected by the investigator, double-labeled with fluorescent reporter and quencher dye molecules. As amplicons accumulate during the course of the reaction, the probe will hybridize to any target sequence present on the amplicons. When exposed to pulses of intense light, the fluorescent dyes located on the probe will respond with characteristic emission spectra which can be presented to the user as amplification plots. Because different probes can be labeled with different dye molecules, it is possible to simultaneously assay the same sample for the presence of different target sequences (i.e., “multiplexing”). In practical terms, this allows the same sample to be assayed for the presence of several different threat agents in a one tube reaction.
Strand Displacement Amplification (SDA): is actually a non-PCR nucleic acid amplification technique developed in 1991. DNA polymerase initiates DNA syntheses at a single stranded nick and displaces the nicked strand during DNA synthesis. The displaced single stranded molecule then serves a a substrate for additional simultaneous nicking and displacement reactions. This isothermal DNA amplification procedure users specific primers, a DNA polymerase, and a restriction endonuclease to achieve exponential amplification of target. The key technology behind SDA is the generation of site specific nicks by the restriction endonuclease.
Touchdown PCR: involves decreasing the annealing temperature by 1 degree C every second cycle to a “touchdown” annealing temperature which is then used for 10 or so cycles.
Transcription Based Amplification System (TAS): includes synthesis of a DNA molecule complementary to the target nucleic acid (usually RNA) and in vitro transcription with newly synthesized cDNA as a template. Variations on this process are referred to as self-sustaining sequence replication (3SR), nucleic acid sequence-based amplification (NASBA) or transcription mediated amplification (TMA) Three enzymes, RT, RNase H, and T7 DNA dependent RNA polymerase are used in the reaction. Amplification steps involve the formation of cDNA from the target RNA by using primers containing a RNA polymerase binding site. The RNase H then degrades the initial strand of target RNA in the RNA-DNA hybrid after it has served as the template for the first primer. The second primer binds to the newly formed cDNA and is extended, resulting in the formation of double-strand cDNAs in which one or both strands are capable of serving as transcription templates for RNA polymerase. Although technically less robust and less sensitive than PCR, TMA has various merits that maike an attractive option: it works at isothermal conditions in a single tube to help minimize contamination risks.