Types of PCR | Daniel Apeh Academia.edu
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refers to reverse transcription PCR (see below), often used in conjunction with QPCR [27]. OTHER TYPES OF PCR LONG PCR
Long PCR is a PCR is which extended or longer than standard PCR, meaning over 5 kilobases (frequently over 10 kb). Long PCR is usually only useful if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together.
Applications of
Long PCR Long PCR is often used to clone larger genes or large segments of
DNA which standard PCR cannot [27]. COLONY PCR
T he screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products [27]. Selected colonies of bacteria or yeast are picked with a sterile toothpick or pipette tip from a growth (agarose) plate. This is then inserted into the PCR master mix or preinserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or data:text/html;charset=utf8,%3Cbr%20class%3D%22Appleinterchangenewline%22%3E%3Cdiv%20class%3D%22outer_page%20only_ie6_border%20%…
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plasmid of interest [28]. THE DIGITAL PCR
The Digital polymerase chain reaction simultaneously amplifies thousands of samples, each in a separate droplet within an emulsion [2
9] .
OVERLAPEXTENSION PCR
A genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length [30]. SOLID PHASE PCR
encompasses multiple meanings, including Colony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (primers are covalently linked to a solidsupport surface), conventional Solid Phase PCR 26
(where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be improved by employing high Tm and nested solid support primer with optional application of a thermal 'step' to favour solid support priming) [31]. TOUCHDOWN PCR (STEPDOWN PCR)
A variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (35°C) above the T
m
of the primers used, while at the later cycles, it is a few degrees (35°C) below the primer T m. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles [32]. MINIPRIMER PCR
This reaction uses a thermostable polymerase (STbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding regions, and is used to amplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene [27]. UNIVERSAL FAST WALKING PCR
Used for genome walking and genetic fingerprinting using a more specific data:text/html;charset=utf8,%3Cbr%20class%3D%22Appleinterchangenewline%22%3E%3Cdiv%20class%3D%22outer_page%20only_ie6_border%20%…
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Used for genome walking and genetic fingerprinting using a more specific 'twosided' PCR than conventional 'onesided' approaches (using only one genespecific primer and one general primer which can lead to artefactual 'noise') by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariatdependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe [27].
VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR 27
This method targets areas of the genome that exhibit length variation. The analysis of the genotypes of the sample usually involves sizing of the amplification products by gel electrophoresis. Analysis of smaller VNTR segments known as Short Tandem Repeats (or STRs) is the basis for DNA Fingerprinting databases such as CODIS [27]. INTERSEQUENCESPECIFIC PCR
(OR ISSRPCR )
This is a method for DNA fingerprinting that uses primers selected from segments repeated throughout a genome to produce a unique fingerprint of amplified product lengths. The use of primers from a commonly repeated segment is called
AluPCR , and can help amplify sequences adjacent (or
between) these repeats [27]. CONCLUSION
Current variations of PCR in use are more in number than those highlighted in this discussion, most of these PCRs have specific applications even in new areas of science. This revive have brought out most types of PCR and detailed some even to specific methodology, their principles of operation and their use and also successfully shown that the possibilities that come with the manipulation of DNA are inexhaustible
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