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Types of PCR ((APEH Daniel O.))


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Types of PCR ((APEH Daniel O.))

  1. 1. Apeh Daniel O. TYPES OF POLYMERASE CHAIN REACTIONDNA Replication which forms the basis of biological evolution and inheritance [1] isa "semi conservative" process in that each (one) strand of the original double-strandedDNA molecule serves as template for the reproduction of the complementary strand.Hence, following DNA replication, two identical DNA molecules are been producedfrom a single double-stranded DNA molecule [2].The need to amplify genes for various purposes among which are forensic application,genome studies, medical applications have led to the development of varioustechniques now known as polymerase chain reaction (PCR) as a more convenientalternative of gene cloning via recombinant DNA technology.The idea of Polymerase chain reaction came up in 1983 when Kary Mullis a scientistworking for Cletus cooperation was driving along US route 101 in NorthernCalifornia; it was then introduced into the scientific community in 1985 at aconference in October where Cetus also rewarded kary Mullis with $10,000 bonus forhis invention. Later, during a corporate reorganization, Cetus sold the patent for thePCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million [3].PCR technique which is also called a DNA photocopier, is an in vitro technique thatuses a few basic everyday molecular biology reagents to make large numbers ofcopies of a specific DNA fragment or a specific region of a DNA strand in a test-tube.The process which is carried out in a PCR machine requires DNA template, primer(s),Taq or other polymerase(s), deoxynucleoside triphosphates (dNTPs), buffer solutionand divalent cations (eg.Mg2+ ) to run [2].The basic steps in conducting a conventional PCR involves; Denaturation achieved byheating the reaction mixture to a temperature between 90-98° C such that the dsDNAis denatured into single strands by disrupting the hydrogen bonds betweencomplementary bases, Annealing achieved by cooling the reaction mixture to atemperature of 45-60° C such that the primers base pair with the complementarysequence in the DNA and the hydrogen bonds reform and Elongation achieved byadjusting the temperature to 72° C which is ideal for polymerase allowing primers 1
  2. 2. extension by joining the bases complementary to DNA strands, the polymerase continually adds dNTPs from 5 to 3, reading the template from 3 to 5 side, bases are added complementary to the template. This completes a first cycle another cycle is continued. As PCR machine is automated thermocycler the same cycle is repeated upto 30-40 times [1]. This review attempts to summarize as many types of PCR as possible including the principles on which they work, their applications and in some cases their advantages and disadvantages as well as experimental procedures where necessary. The emphasis is neither placed on the PCR machine level of sophiscation nor time of its use (old or new) but on technical difference basically brought about by different applications of PCR. TYPES OF POLYMERASE CHAIN REACTION INVERSE PCR The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences [4]. Inverse PCR Steps1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).4) Standard PCR is conducted with primers complementary to the now known internal sequences. 2
  3. 3. Inverse PCR uses standard PCR (polymerase chain reaction), however it has theprimers oriented in the reverse direction of the usual orientation. The template for thereverse primers is a restriction fragment that has been ligated upon itself to form acircle [5].Figure 1.0. Inverse PCR ProtocolUses: It is commonly used to identify the flanking sequences around genomic inserts.Inverse PCR has numerous applications in molecular biology including theamplification and identification of sequences flanking transposable elements, and theidentification of genomic inserts [6]. 3
  4. 4. MULTIPLEX PCR Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis [7]. Uses : Its has been found useful in Pathogen Identification, High Throughput SNP Genotyping, Mutation Analysis, Gene Deletion Analysis, Template Quantification, Linkage Analysis, RNA Detection and Forensic Studies [8]. Types of Multiplex PCR Multiplexing reactions can be broadly divided into two1. Single template PCR reaction; this technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template2. Multiple template PCR reaction; this technique uses multiple templates and several primer sets in the same reaction tube. Presence of multiple primer may lead to cross hybridization with each other and the possibility of mis-priming with other templates. 4
  5. 5. Figure 2.0 Primer Design Parameters for Multiplex PCR Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations described below are a key to specific amplification with high yield [9].1. Primer Length: Multiplex PCR assays involve designing of large number of primers, hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used.2. Melting Temperature: Primers with similar Tm, preferably between 55°C-60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3°-5° C is acceptable for primers used in a pool. 5
  6. 6. 3. Specificity: It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel.4. Avoid Primer Dimer Formation: The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification. All other parameters are similar to standard PCR primer design guidelines. Advantages of Multiplex PCR1. Internal Controls: Potential problems in a simple PCR include false negatives due to reaction failure or false positives due to contamination. False negatives are often revealed in multiplex assays because each amplicon provides an internal control for the other amplified fragments.2. Efficiency: The expense of reagents and preparation time is less in multiplex PCR than in systems where several tubes of uniplex PCRs are used. A multiplex reaction is ideal for conserving costly polymerase and templates in short supply.3. Indication of Template Quality: The quality of the template may be determined more effectively in multiplex than in a simple PCR reaction.4. Indication of Template Quantity: The exponential amplification and internal standards of multiplex PCR can be used to assess the amount of a particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of reference template, the number of reaction cycles, and the minimum inhibition of the theoretical doubling of product for each cycle must be accounted. 6
  7. 7. NESTED PCRThis PCR increases the specificity of DNA amplification, by reducing backgrounddue to non-specific amplification of DNA [10]. Two sets (instead of one pair) ofprimers are used in two successive PCRs. In the first reaction, on pair of primers“outer pair” is used to generate DNA products, which besides the intended target, maystill consist of non-specifically amplified DNA fragments. The product(s) are thenused in a second PCR after the reaction is diluted with a set of second set “nested orinternal” primers whose binding sites are completely or partially different from andlocated 3 of each of the primers used in the first reaction. The specificity of PCR isdetermined by the specificity of the PCR primers. For example, if your primers bindto more than one locus (e.g. paralog or common domain), then more than one segmentof DNA will be amplified. To control for these possibilities, investigators oftenemploy nested primers to ensure specificity [11].Nested PCR means that two pairs of PCR primers were used for a single locus (figure1). The first pair amplified the locus as seen in any PCR experiment. The second pairof primers (nested primers) bind within the first PCR product (figure 4) and producea second PCR product that will be shorter than the first one (figure 5). The logicbehind this strategy is that if the wrong locus were amplified by mistake, theprobability is very low that it would also be amplified a second time by a second pairof primers [12]. 7
  8. 8. Figure 3.0 Nested PCR patternFigure 3.1. Nested PCR strategy. Segment of DNA with dots representing nondiscriptDNA sequence of unspecified length. The double lines represent a large distancebetween the portion of DNA illustrated in this figure. The portions of DNA shownwith four bases in a row represent PCR primer binding sites, though real primerswould be longer.Figure 3.2. The first pair of PCR primers (blue with arrows) bind to the outer pair ofprimer binding sites and amplify all the DNA in between these two sites. 8
  9. 9. Figure 3.3. PCR product after the first round of amplificaiton. Notice that the basesoutside the PCR primer pair are not present in the product.Figure 3.4. Second pair of nested primers (red with arrows) bind to the first PCRproduct. The binding sites for the second pair of primers are a few bases "internal" tothe first primer binding sites.Figure 3.5. Final PCR product after second round of PCR. The length of the productis defined by the location of the internal primer binding sites.When a complete genome sequence is known, it is easier to be sure you will notamplify the wrong locus but since very few of the worlds genomes have beensequenced completely, nested primers will continue to be an important control formany experiments.A drawback of this technique is that the addition of new primers after the firstamplification round increases the chances of nonspecific contamination; many clinicallabs avoid this technique for this reason [11]. 9
  10. 10. MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA)MLPA is used to establish the copy number of up to 45 nucleic acid sequences in onesingle multiplex reaction. The method can be used for genomic DNA (including bothcopy number detection and methylation quantification) as well as for mRNAprofiling, it permits multiple targets to be amplified with only a single primer pair,thus avoiding the resolution limitations of multiplex PCR [13].The principle of MLPA is based on the identification of target sequences byhybridization of pairs of MLPA probes that bind to adjacent sequences and can thenbe joined by a ligation reaction. In order to make one copy of each target sequence,specific MLPA probes are added to a nucleic acid sample for each of the sequences ofinterest.The sequences are then simultaneously amplified with the use of only one primer pair,resulting in a mixture of amplification products, in which each PCR product of eachMLPA probe has a unique length [13].One PCR primer is fluorescently or isotopically labelled so that the MLPA reactionproducts can be visualized when electrophoresed on a capillary sequencer or a gel.Resulting chromatograms show size-separated fragments ranging from 130 to 490 bp.The peak area or peak height of each amplification product reflects the relative copynumber of that target sequence. Comparison of the electrophoresis profile of thetested sample to that obtained with a control sample enables the detection of deletionsor duplications of genomic regions of interest [14].Figure 4.0 summarizes the MLPA workflow for common devices and tools for dataanalysis 10
  11. 11. 11Figure 4.0. MLPA workflow for common devices and tools for data analysis
  12. 12. LIGATION-MEDIATED PCRLigation-mediated PCR uses small DNA oligonucleotide linkers (or adaptors)that are first ligated to fragments of the target DNA. PCR primers that annealto the linker sequences are then used to amplify the target fragments. Thismethod is deployed for DNA sequencing, genome walking, and DNAfootprinting A related technique is Amplified fragment length polymorphism,which generates diagnostic fragments of a genome [15].Primer-extension step (Step 3): a gene-specific primer (Primer 1) was annealedat 48°C and the primer was extended with Sequenase enzyme at 48°C. Ligationstep (Step 4): all extended DNA fragments with a blunt-end and 5-phosphategroup were ligated to an unphosphorylated synthetic asymmetric double-strandlinker. Linear amplification step (Step 5): a second gene-specific primer(Primer 2) was annealed to DNA fragments for a one-cycle extension usingTaq DNA polymerase. Exponential amplification step (Step 6): the primer 2and the linker primer (the longest of the two oligonucleotides of the linker)were used to exponentially and specifically amplify DNA fragments.Sequencing gel electrophoresis and electroblotting (Step 7): amplified DNAfragments were size-separated on a denaturing 8% polyacrylamide gel andtransferred onto a nylon membrane by electroblotting. Hybridization (Step 8)the nylon membrane was hybridized overnight with a gene-specific probe. 12
  13. 13. Figure 5.0 Ligation-Mediated PCR flow setup 13
  14. 14. The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess ofprimers, 2-Polymerase chain reaction of individual fragments. In LM-PCR,each fragment is amplified independently so that due to intrinsic differencesamong individual fragments, some fragments are amplified less efficiently thanothers. This results in non-uniform representation of original genetic materialin the resultant amplicon, which consequently leads to loss of geneticinformation and inaccurate results [16].Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitivesequencing technique available to map single-stranded DNA breaks at thenucleotide level of resolution using genomic DNA. LMPCR has been adaptedto map DNA damage and reveal DNA–protein interactions inside living cells.However, the sequence context (GC content), the global break frequency andthe current combination of DNA polymerases used in LMPCR affect thequality of the results. In this study, we developed and optimized an LMPCRprotocol adapted for Pyrococcus furiosus exo– DNA polymerase (Pfu exo–).The relative efficiency of Pfu exo– was compared to T7-modified DNApolymerase (Sequenase 2.0) at the primer extension step and to Thermusaquaticus DNA polymerase (Taq) at the PCR amplification step of LMPCR.At all break frequencies tested, Pfu exo– proved to be more efficient thanSequenase 2.0. During both primer extension and PCR amplification steps, theratio of DNA molecules per unit of DNA polymerase was the maindeterminant of the efficiency of Pfu exo–, while the efficiency of Taq was lessaffected by this ratio. Substitution of NaCl for KCl in the PCR reaction bufferof Taq strikingly improved the efficiency of the DNA polymerase. Pfu exo–was clearly more efficient than Taq to specifically amplify extremely GC-richgenomic DNA sequences. Our results show that a combination of Pfu exo– atthe primer extension step and Taq at the PCR amplification step is ideal for invivo DNA analysis and DNA damage mapping using LMPCR [17]. 14
  15. 15. METHYLATION-SPECIFIC PCR (MSP)Methylation-specific PCR (MSP) is used to identify patterns of DNAmethylation at cytosine-guanine (CpG) islands in genomic DNA [18]. TargetDNA is first treated with sodium bisulphite, which converts unmethylatedcytosine bases to uracil, which is complementary to adenosine in PCR primers.Two amplifications are then carried out on the bisulphite-treated DNA: Oneprimer set anneals to DNA with cytosines (corresponding to methylatedcytosine), and the other set anneals to DNA with uracil (corresponding tounmethylated cytosine). MSP used in Q-PCR provides quantitative informationabout the methylation state of a given CpG island [18].Bisulphite sequencing (also known as bisulphite sequencing) is the use ofbisulfite treatment of DNA to determine its pattern of methylation. DNAmethylation was the first discovered epigenetic mark, and remains the moststudied. In animals it predominantly involves the addition of a methyl group tothe carbon-5 position of cytosine residues of the dinucleotide CpG, and isimplicated in repression of transcriptional activity.Treatment of DNA with bisulphite converts cytosine residues to uracil, butleaves 5-methylcytosine residues unaffected. Thus, bisulphite treatmentintroduces specific changes in the DNA sequence that depend on themethylation status of individual cytosine residues, yielding single- nucleotideresolution information about the methylation status of a segment of DNA.Various analyses can be performed on the altered sequence to retrieve thisinformation. The objective of this analysis is therefore reduced todifferentiating between single nucleotide polymorphisms (cytosines andthymidine) resulting from bisulphite conversion. 15
  16. 16. Figure 6.0 Outline of the chemical reaction that underlies the bisulphite-mediated conversion of cytosine to uracil.Figure 6.1 Methylation-specific PCR flowMethylation-specific PCR is a sensitive method to discriminately amplify anddetect a methylated region of interest using methylated-specific primers onbisulfite-converted genomic DNA. Such primers will anneal only to sequencesthat are methylated, and thus containing 5-methylcytosines that are resistant toconversion by bisulfite. In alternative fashion, unmethylated-specific primerscan be used. This alternative method of methylation analysis also usesbisulfite-treated DNA but avoids the need to sequence the area of interest.Instead, primer pairs are designed themselves to be "methylated-specific" by 16
  17. 17. including sequences complementing only unconverted 5-methylcytosines, or,on the converse, "unmethylated-specific", complementing thymines convertedfrom unmethylated cytosines.Methylation is determined by the ability of the specific primer to achieveamplification. This method is particularly useful to interrogate CpG islandswith possibly high methylation density, as increased numbers of CpG pairs inthe primer increase the specificity of the assay. Placing the CpG pair at the 3-end of the primer also improves the sensitivity. The initial report using MSPdescribed sufficient sensitivity to detect methylation of 0.1% of alleles. Ingeneral, MSP and its related protocols are considered to be the most sensitivewhen interrogating the methylation status at a specific locus.The MethyLight method is based on MSP, but provides a quantitative analysisusing real-time PCR. Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplifiedregion. In alternative fashion, the primers or probe can be designed withoutmethylation specificity if discrimination is needed between the CpG pairswithin the involved sequences. Quantitation is made in reference to amethylated reference DNA. A modification to this protocol to increase thespecificity of the PCR for successfully bisulphite-converted DNA (ConLight-MSP) uses an additional probe to bisulphite-unconverted DNA to quantify thisnon-specific amplification [19].Further methodology using MSP-amplified DNA analyzes the products usingmelting curve analysis (Mc-MSP).This method amplifies bisulphite-convertedDNA with both methylated-specific and unmethylated-specific primers, anddetermines the quantitative ratio of the two products by comparing thedifferential peaks generated in a melting curve analysis. A high-resolutionmelting analysis method that uses both real-time quantification and meltinganalysis has been introduced, in particular, for sensitive detection of low-levelmethylation [20] . 17
  18. 18. HOT-START PCRA technique that reduces non-specific amplification during the initial set upstages of the PCR. It may be performed manually by heating the reactioncomponents to the melting temperature (e.g., 95°C) before adding thepolymerase. Specialized enzyme systems have been developed that inhibit thepolymerases activity at ambient temperature, either by the binding of anantibody or by the presence of covalently bound inhibitors that only dissociateafter a high-temperature activation step. Hot-start/cold-finish PCR is achievedwith new hybrid polymerases that are inactive at ambient temperature and areinstantly activated at elongation temperature [21].Mechanical hot start PCR: all components of PCR are added to the PCR vialexcept for the DNA polymerase enzyme which will be added just at the firstdenaturation step.Non mechanical hot start PCR: The use of a form of Taq DNA polymerase,for example, Amplitaq Gold which is activated only if the reaction mixture isheated at about 94°C (the first denaturation step). Other method depends oncovalent linking of the polymerase enzyme to certain inhibitors. The enzymebecomes dissociated from these inhibitors at the first denaturation step. Figure 7.0 Hot-Start PCR flow 18
  19. 19. ALLELE-SPECIFIC PCRA diagnostic or cloning technique which is based on single-nucleotidepolymorphisms (SNPs) (single-base differences in DNA). It requires priorknowledge of a DNA sequence, including differences between alleles, and usesprimers whose 3 ends encompass the SNP. PCR amplification under stringentconditions is much less efficient in the presence of a mismatch betweentemplate and primer, so successful amplification with an SNP-specific primersignals presence of the specific SNP in a sequence [22].Figure 8.0 Allele-Specific PCR flowHELICASE-DEPENDENT AMPLIFICATIONThis PCR is similar to traditional PCR, but uses a constant temperature ratherthan cycling through denaturation and annealing/extension cycles. DNAhelicase, an enzyme that unwinds DNA, is used in place of thermaldenaturation [27]. 19
  20. 20. REVERSE TRANSCRIPTION PCR (RT-PCR)A PCR designed for amplifying DNA from RNA. Reverse transcriptasereverse transcribes RNA into cDNA, which is then amplified by PCR. RT-PCRis widely used in expression profiling, to determine the expression of a gene orto identify the sequence of an RNA transcript, including transcription start andtermination sites. If the genomic DNA sequence of a gene is known, RT-PCRcan be used to map the location of exons and introns in the gene. The 5 end ofa gene (corresponding to the transcription start site) is typically identified byRACE-PCR (Rapid Amplification of cDNA Ends) [23].Figure 9.0 Reverse Transcription PCR flowIN SITU PCR (ISH)A polymerase chain reaction that actually takes place inside the cell on a slide.In situ PCR amplification can be performed on fixed tissue or cells [24]. 20
  21. 21. ASSEMBLY PCR OR POLYMERASE CYCLING ASSEMBLY (PCA)This entails the artificial synthesis of long DNA sequences by performing PCRon a pool of long oligonucleotides with short overlapping segments. Theoligonucleotides alternate between sense and antisense directions, and theoverlapping segments determine the order of the PCR fragments, therebyselectively producing the final long DNA product [25].ASYMMETRIC PCRThis reaction preferentially amplifies one DNA strand in a double-strandedDNA template. It is used in sequencing and hybridization probing whereamplification of only one of the two complementary strands is required. PCR iscarried out as usual, but with a great excess of the primer for the strandtargeted for amplification. Because of the slow (arithmetic) amplification laterin the reaction after the limiting primer has been used up, extra cycles of PCRare required. A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a highermelting temperature (Tm) than the excess primer to maintain reactionefficiency as the limiting primer concentration decreases mid-reaction [26].THERMAL ASYMMETRIC INTERLACED PCR (TAIL-PCR)This reaction is applied in the isolation of an unknown sequence flanking aknown sequence. Within the known sequence, TAIL-PCR uses a nested pair ofprimers with differing annealing temperatures; a degenerate primer is used toamplify in the other direction from the unknown sequence [27].Uses: TAIL-PCR as a powerful tool for amplifying insert end segments fromP1, BAC and YAC clones, the amplified products were highly specific andsuitable as probes for library screening and as templates for direct sequencing 21
  22. 22. while the recover insert ends can also be used for chromosome walking andmappingThermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is afast and efficient method to amplify unknown sequences adjacent to knowninsertion sites in Arabidopsis. Nested, insertion-specific primers are usedtogether with arbitrary degenerate primers (AD primers), which are designed todiffer in their annealing temperatures. Alternating cycles of high and lowannealing temperature yield specific products bordered by an insertion-specificprimer on one side and an AD primer on the other. Further specificity isobtained through subsequent rounds of TAIL-PCR, using nested insertion-specific primers. The increasing availability of whole genome sequencesrenders TAIL-PCR an attractive tool to easily identify insertion sites in largegenome tagging populations through the direct sequencing of TAIL-PCRproducts. For large-scale functional genomics approaches, it is desirable toobtain flanking sequences for each individual in the population in a fast andcost-effective manner.Experimental Details Primary reaction.In the primary reaction, one low stringency PCR cycle is conducted to createone or more annealing sites for the AD primer in the targeted sequence.Specific products are then amplified over non-specific ones by interspersion oftwo high-stringency PCR cycles with one reduced-stringency PCR cycle.Set up 4 reactions as follows (one with each AD primer):2 μl 10 X PCR buffer1.2 μl 25 mM MgCl20.2 μl 10 mMdNTP’s0.2 μl 100 ngμl-1 specific primer 1 (furthest away from AD) (0.15μM final)2 μl20 μM AD primer (2 μM final) 22
  23. 23. 0.2 μlTaq DNA polymerase0.4 μl DMSO1 μl DNA (1-20ngμl-1)12.8 μlH2OThere is no need to run out this primary reaction. It should contain a mediumyield of specific products, a high yield of non-targeted products, and a lowyield of non-specific products. The nested primers used in the secondary andtertiary reactions result in very low yields of non-specific products, very highyields of specific products and no amplification of non-targeted products. Secondary reactionFor the secondary reaction, a 1/40 dilution of the primary PCR product is usedas template, and the specific primer is the middle one of the three specificprimers.Set up reaction as follows:2.5 μl 10 X PCR buffer1.5 μl 25 mM MgCl20.25 μl 10 mMdNTP’s0.3 μl 100 ngμl-1 specific primer 2 (middle nested) (0.2 μM final)2.5 μl 20 μM AD primer (2 μM final)0.2 μl Taq DNA polymerase0.5 μl DMSO1 μl DNA (1/40 dilution of primary PCR products)16.25 μl H2O Tertiary reactionFor the tertiary reaction, the SAME template (i.e primary PCR product) is usedbut thistime in a 1/10 dilution. I usually simply add 4 X of the 1/40 dilutionused for thesecondary reaction. This removes the possibility of getting falsepositives. The specificprimer used is the primer nearest the unknown sequence. 23
  24. 24. Set up reaction as follows:5 μl10 X PCR buffer3 μl25 mM MgCl20.5 μl10 mMdNTP’s0.6 μl 100ngμl-1 specific primer 3 (closest to AD) (0.2 μM final)5 μl20 μM any one AD primer (2 μM final)0.4 μlTaq DNA polymerase1 μl DMSO4 μlDNA (1/40 dilution of primary PCR products)31 μl H2OAgarose gel analysisThe secondary and tertiary products are run in adjacent lanes on a 1.2%agarose gel. The specificity of the products is confirmed by the expected sizechange between the secondary and tertiary products. 24
  25. 25. QUANTITATIVE PCR (Q-PCR)Used to measure the quantity of a PCR product (commonly in real-time). Itquantitatively measures starting amounts of DNA, cDNA or RNA. Q-PCR iscommonly used to determine whether a DNA sequence is present in a sampleand the number of its copies in the sample. Quantitative real-time PCR has avery high degree of precision. QRT-PCR methods use fluorescent dyes, suchas Sybr Green, EvaGreen or fluorophore-containing DNA probes, such asTaqMan, to measure the amount of amplified product in real time. It is alsosometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCRor RTQ-PCR are more appropriate contractions, since RT-PCR commonly 25
  26. 26. refers to reverse transcription PCR (see below), often used in conjunction withQ-PCR [27].OTHER TYPES OF PCRLONG PCRLong PCR is a PCR is which extended or longer than standard PCR, meaningover 5 kilobases (frequently over 10 kb). Long PCR is usually only useful if itis accurate. Thus, special mixtures of proficient polymerases along withaccurate polymerases such as Pfu are often mixed together. Applications ofLong PCR Long PCR is often used to clone larger genes or large segments ofDNA which standard PCR cannot [27].COLONY PCRThe screening of bacterial (E.Coli) or yeast clones for correct ligation orplasmid products [27]. Selected colonies of bacteria or yeast are picked with asterile toothpick or pipette tip from a growth (agarose) plate. This is theninserted into the PCR master mix or pre-inserted into autoclaved water. PCRis then conducted to determine if the colony contains the DNA fragment orplasmid of interest [28].THE DIGITAL PCRThe Digital polymerase chain reaction simultaneously amplifies thousands ofsamples, each in a separate droplet within an emulsion [29].OVERLAP-EXTENSION PCRA genetic engineering technique allowing the construction of a DNA sequencewith an alteration inserted beyond the limit of the longest practical primerlength [30].SOLID PHASE PCRencompasses multiple meanings, including Colony Amplification (where PCRcolonies are derived in a gel matrix, for example), Bridge PCR (primers arecovalently linked to a solid-support surface), conventional Solid Phase PCR 26
  27. 27. (where Asymmetric PCR is applied in the presence of solid support bearingprimer with sequence matching one of the aqueous primers) and EnhancedSolid Phase PCR (where conventional Solid Phase PCR can be improved byemploying high Tm and nested solid support primer with optional applicationof a thermal step to favour solid support priming) [31].TOUCHDOWN PCR (STEP-DOWN PCR)A variant of PCR that aims to reduce nonspecific background by graduallylowering the annealing temperature as PCR cycling progresses. The annealingtemperature at the initial cycles is usually a few degrees (3-5°C) above the T mof the primers used, while at the later cycles, it is a few degrees (3-5°C) belowthe primer Tm. The higher temperatures give greater specificity for primerbinding, and the lower temperatures permit more efficient amplification fromthe specific products formed during the initial cycles [32].MINIPRIMER PCRThis reaction uses a thermostable polymerase (S-Tbr) that can extend fromshort primers ("smalligos") as short as 9 or 10 nucleotides. This methodpermits PCR targeting to smaller primer binding regions, and is used toamplify conserved DNA sequences, such as the 16S (or eukaryotic 18S) rRNAgene [27].UNIVERSAL FAST WALKING PCRUsed for genome walking and genetic fingerprinting using a more specifictwo-sided PCR than conventional one-sided approaches (using only onegene-specific primer and one general primer - which can lead to artefactualnoise) by virtue of a mechanism involving lariat structure formation.Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nestedPCR for rapid amplification of genomic DNA ends), 5RACE LaNe and3RACE LaNe [27].VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR 27
  28. 28. This method targets areas of the genome that exhibit length variation. Theanalysis of the genotypes of the sample usually involves sizing of theamplification products by gel electrophoresis. Analysis of smaller VNTRsegments known as Short Tandem Repeats (or STRs) is the basis for DNAFingerprinting databases such as CODIS [27].INTERSEQUENCE-SPECIFIC PCR (OR ISSR-PCR)This is a method for DNA fingerprinting that uses primers selected fromsegments repeated throughout a genome to produce a unique fingerprint ofamplified product lengths. The use of primers from a commonly repeatedsegment is called Alu-PCR, and can help amplify sequences adjacent (orbetween) these repeats [27].CONCLUSIONCurrent variations of PCR in use are more in number than those highlighted in thisdiscussion, most of these PCRs have specific applications even in new areas ofscience. This revive have brought out most types of PCR and detailed some even tospecific methodology, their principles of operation and their use and also successfullyshown that the possibilities that come with the manipulation of DNA are inexhaustible..REFERENCES 28
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