Asymmetric PCR: A PCR in which the predominant product is a single-stranded DNA, as a result of unequal primer concentrations.   As asymmetric PCR proceeds, the lower concentration primer is quantitatively incorporated into double-stranded DNA.  The higher concentration primer continues to primer synthesis, but only of its strand.

 

cDNA synthesis (aka reverse transcription or RT):  cDNA is a DNA copy synthesized from mRNA.  The enzyme used is reverse transcriptase, an RNA-dependent DNA polymerase isolated from a retrovirus (AMV or MMLV).  As with other DNA polymerases a short double-stranded sequence is needed at the 3' end of the mRNA which acts as a start point for the polymerase.  This is provided by the poly(A) tail found at the 3' end of most eukaryotic mRNAs to which a short complementary synthetic oligonucleotide (oligo dT primer) is hybridized (polyT-polyA hybrid).   Together with all 4 deoxynucleoside triphosphates, magnesium ions and at neutral pH, the reverse transcriptase synthesizes a complementary DNA on the mRNA template. (http://www.dur.ac.uk/biological.sciences/Staff/Croy/cDNAfigs.htm)

 

DNA mobility shift assay: The electrophoretic mobility shift assay (EMSA) is often used to examine DNA-binding proteins (Reference: http://www.fgsc.net/fgn45/45meyer.html).

(Reference: http://microimm.queensu.ca/micr436/notes/436-geneexpression/tsld021.htm)

Hybridization:  This term refers to the formation of a double-stranded nucleic acid structure from two single strands that were not previously associated.  Because formation of the double helix depends on accurate base pairing, hybridization is used in detection of nucleic acid sequences.  One strand is called the “probe” while the other is the “target” (usually in the sample to be assayed).

 

Dot-blot Hybridization: dot blot is a technique for immobilizing several preparations of nucleic acids on the same solid support, usually a charged nylon membrane.  The concentrations of the target sequence of interest can be estimated by hybridizing the immobilized samples to an appropriate probe. (1)

 

In situ hybridization: In situ hybridization, as the name suggests, is a method of localizing, either mRNA within the cytoplasm or DNA within the chromosomes of the nucleus, by hybridizing the sequence of interest to a complementary strand of a poly- or oligo-nucleotide probe. (Reference: http://martin.parasitology.mcgill.ca/insituhybridization/insitu.htm#Introduction)

 

Northern and Southern hybridization refer to the probing, by hybridization, of a membrane (nylon or nitrocellulose) containing RNA or DNA, respectively, which has been transferred from a gel electrophoretic separation by blotting.

 

Inverse PCR: Standard PCR amplifies segments of DNA that lie between two inward-pointing primers.  By contrast, inverse (also known as inverted or inside-out) PCR is used to amplify and clone unknown DNA that flanks one end of a known DNA sequence and for which no primers are available.  The technique involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region.  The individual restriction fragments are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR.  The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite direction. (1)

Multiplex PCR: Multiplex PCR is the term used when more than one pair of primers is used in a PCR.  The goal of multiplex PCR is to amplify several segments of target DNA simultaneously and thereby to conserve template DNA, save time, and minimize expense (1).   It is a PCR strategy that enables the amplification of multiple DNA targets in one run.  This PCR technique is used for genetic screening, microsatellite analysis, and other applications where it is necessary to amplify several products in a single reaction. This technique often requires extensive optimization because having multiple primer pairs in a single reaction increases the likelihood of primer–dimers and other nonspecific products that may interfere with the amplification of specific products. In addition, the concentrations of individual primer pairs often need to be optimized since different multiplex amplicons are often amplified with differing efficiencies, and multiple primer pairs can compete with each other in the reaction.  (http://www.qiagen.com/clinical/applications/technologies/multiplex_pcr.asp)

Multiplex RT-PCR:  Multiplex RT-PCR (also referred to as relative RT-PCR) is commonly used for the semi-quantitative analysis of gene expression levels when defining tissue-restricted gene expression patterns. Typically, multiplex RT-PCR is performed to determine the changes in expression level of a gene in a series of tissue types, throughout stages of development or cellular differentiation, or after specific experimental treatments. Multiplex RT-PCR is also commonly used to examine the expression patterns of a series of related genes and to look at various regions of a large message for mutation analysis. (Reference: http://www.epicentre.com/f5_4rtpcrmulti.asp)

Multiplex RT-PCR is a time and reagent saving amplification technique in which multiple primer sets are used to amplify multiple specific targets simultaneously from the same sample. (Reference: http://www.westburg.nl/htm/products/pcr_and_rtpcr/rotorgene.htm)

 

NASBA: It stands for nucleic acid sequence-based amplification.  NASBA is a transcription-based amplification method which amplifies RNA from either an RNA or DNA target. (Reference: http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-N/NASBA.html)

 

Nested PCR: Nested PCR refers to a pair of PCRs run in series each with a pair of primers flanking the same sequence. The first PCR amplifies a sequence as seen in any PCR experiment. The second pair of primers (nested primers) for the second PCR bind within the first PCR product and produce a second PCR product that is shorter than the first one. The technique, because it uses four specific primers, rather than two, has greater specificity than regular PCR.  It can also yield detectable product in cases where simple PCR fails to do so.  (Reference: http://www.bio.davidson.edu/courses/genomics/method/NestedPCR.html)

Nested RT-PCR: This term refers to a nested PCR reaction that is initiated with cDNA that has been reverse transcribed from RNA.

 

PCR: The polymerase chain reaction is a test tube system for DNA replication that allows a "target" DNA sequence to be selectively amplified, or enriched, several million-fold in just a few hours. Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions, whose end result is a faithful copy of the entire genome. Within a test tube, PCR uses just one indispensable enzyme - DNA polymerase - to amplify a specific fraction of the genome. (http://www.accessexcellence.com/RC/CT/polymerase_chain_reaction.html)

Primer extension: Primer extension is used to map the 5' ends of DNA or RNA fragments.  It is done by annealing a specific oligonucleotide primer to a position downstream of that 5' end. The primer is labeled, usually at its 5' end, with 32P. This is extended with reverse transcriptase, which can copy either an RNA or a DNA template, making a fragment that ends at the 5' end of the template molecule. DNA polymerase can also be used with DNA templates. (Reference: http://www.biochem.arizona.edu/classes/bioc568/primer_extension.htm)

Q-RT-PCR:  It stands for quantitative reverse transcription-PCR.  An early method of  QRT-PCR involves comparing the amount of specific product generated in different samples from a particular target sequence.  In this technique, the amounts of amplified product were measured at several time points during the exponential phase of the reaction and were analyzed by linear regression.  (1)

 

Real-time PCR: Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production during each PCR cycle (i.e., in real time) as opposed to the endpoint detection by conventional quantitative PCR methods. The real-time PCR system is based on the detection and quantitation of a fluorescent reporter. This signal increases in direct proportion to the amount of PCR product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR reaction during exponential phase where the first significant increase in the amount of PCR product correlates to the initial amount of target template.  There are several strategies for detecting the accumulation of product.  They have in common that a third oligonucleotide bearing fluorescent moieties is required and is complementary to a section in the amplified target.  In Taqman PCR, the fluorescent moiety and a quencher are near one end of the molecule.  Hydrolysis by the advancing polymerase releases the fluorescent nucleotide from the effect of the quencher.  In Molecular Beacons, the fluorophore and the quencher, attached to opposite ends of the oligonucleotide, are held together by a base paired stem that becomes disrupted on hybridization of the loop to a target nucleic acid. (http://dorakmt.tripod.com/genetics/realtime.html)

 

Real-time RT-PCR: This term refers to a real-time PCR that is initiated with cDNA that has been reverse transcribed from RNA.

 

RT-Asymmetric PCR: This term refers to an asymmetric PCR that is initiated with cDNA that has been reverse transcribed from RNA.

 

RT semi-nested PCR: This term refers to a semi-nested PCR that is initiated with cDNA that has been reverse transcribed from RNA.

 

RT-Nested Multiplex PCR: This term refers to a nested PCR that is initiated with cDNA that has been reverse transcribed from RNA and includes multiple primer pairs at one or both of the consecutive PCRs.

Reverse Transcription PCR (RT-PCR): See “cDNA synthesis”.  (1)

 

Semi-nested PCR: Similar to a nested PCR (which see) except that in the second PCR one of the primers is a primer that was used in the first PCR.

 

Universal PCR:  This term refers to PCR using very highly degenerate primers with short variable 3' anchor sequences and 5' adaptors.  The method results in the amplification of many random segments of the target genome.  The adaptor sequences may contain restriction sites to facilitate cloning or may themselves be targets for amplification.  (http://www.sigmaaldrich.com/B2B/Area_of_Interest/Life_Science/Molecular_Biology/Protein_Expression/Cloning_and_Expression/Director_Universal_PCR_System.html)

 

Universal RT-PCR: This term refers to a universal PCR that is initiated with cDNA that has been reverse transcribed from RNA.

 

 

 

Reference:

 

  1. Sambrook J. and D. Russell, Molecular Cloning A Laboratory Manual, 3rd Edition.  Cold Spring Harbor Laboratory Press, 2001.