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DIFFERENT PCR TECHNIQUES AND

THEIR APPLICATIONS

Presented by:Saurabh PandeyPALB-3252

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What is PCR

• It is a molecular technology aim to amplify a single or few copies of the DNA to thousands or millions of copies. • Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include diagnosis of infectious diseases, DNA sequencing and DNA-based phylogeny. • In 1993, Mullis was awarded the Nobel prize in Chemistry along with Michael Smith for his work on PCR.

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Applications of PCR

Molecular Identification Sequencing Genetic Engineering• Molecular Archaeology• Molecular Epidemiology• Molecular Ecology• DNA fingerprinting• Classification of organisms• Genotyping• Pre-natal diagnosis• Mutation screening• Drug discovery• Genetic matching• Detection of pathogens

• Bioinformatics• Genomic cloning• Human Genome Project

• Site-directed mutagenesis• Gene expression studies

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TYPES OF POLYMERASE CHAIN REACTION

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ALLELE-SPECIFIC PCR

•A diagnostic or cloning technique which is based on single-nucleotide polymorphisms (SNPs) (single-base differences in DNA). • It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. • PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.

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Allele-Specific PCR flow

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ASYMMETRIC PCR

• This reaction preferentially amplifies one DNA strand in a double-stranded DNA template.• It is used in sequencing and hybridization probing where amplification of only one of the two complementary strands is required.• PCR is carried out as usual, but with a great excess of the primer for the strand targeted for amplification. •Because of the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required. •A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction .

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ASSEMBLY PCR OR POLYMERASE CYCLING ASSEMBLY (PCA)

• This entails the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. • The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments, thereby selectively producing the final long DNA product.

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Assembly PCR:

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HOT-START PCR

•A technique that reduces non-specific amplification during the initial set up stages of the PCR. • It may be performed manually by heating the reaction components to the melting temperature (e.g., 95°C) before adding the polymerase. • Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. •Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

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•Mechanical hot start PCR: all components of PCR are added to the PCR vial except for the DNA polymerase enzyme which will be added just at the first denaturation 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 is heated at about 94°C (the first denaturation step). Other method depends on covalent linking of the polymerase enzyme to certain inhibitors. The enzyme becomes dissociated from these inhibitors at the first denaturation step.

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Hot-Start PCR flow

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HELICASE-DEPENDENT AMPLIFICATION

•This PCR is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles.• DNA helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.

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IN 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.•UsesDetection and diagnosis of viruses and other infectious agents in specific cell types within tissues.Detection and characterization of tumor cells within tissue.Detection and diagnosis of genetic mutations in inherited diseases.Detection of gene and gene expression in a tissue.Any assay in which identification of cells expressing a target gene is required. Main advantages are low background, high specificity, fast assay with shorter turn-around time and no need of radioactive chemicals.

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 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.• Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle.

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Inverse PCR Steps

• Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.• Self-ligation is induced under low concentrations

causing the phosphate backbone to reform. This gives a circular DNA ligation product.• 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).• Standard PCR is conducted with primers

complementary to the now known internal sequences.

04/09/2023 20Figure: Inverse PCR Protocol

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USES OF INVERSE PCR

• It is commonly used to identify the flanking sequences around genomic inserts.• Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences, flanking transposable elements, and the identification of genomic inserts.

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LIGATION-MEDIATED PCR• Ligation-mediated PCR uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments of the target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target fragments. This method is deployed for DNA sequencing, genome walking, and DNA footprinting.• The principle of Ligation Mediated PCR (LM-PCR). 1-Ligation with excess of primers, 2-Polymerase chain reaction of individual fragments.

In LM-PCR, each fragment is amplified independently so that due to intrinsic differences among individual fragments, some fragments are amplified less efficiently than others. This results in non-uniform representation of original genetic material in the resultant amplicon, which consequently leads to loss of

genetic information and inaccurate results.

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• Primer-extension step (Step 3): a gene-specific primer (Primer 1) was annealed at 48°C and the primer was extended with Sequenase enzyme at 48°C.• Ligation step (Step 4): all extended DNA fragments with a blunt-end and 5'-phosphate group were ligated to an unphosphorylated synthetic asymmetric double-strand linker.• Linear amplification step (Step 5): a second gene-specific primer (Primer 2) was annealed to DNA fragments for a one-cycle extension using Taq DNA polymerase.

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•Exponential amplification step (Step 6): the primer 2 and 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 DNA fragments were size-separated on a denaturing 8% polyacrylamide gel and transferred onto a nylon membrane by electroblotting. •Hybridization (Step 8) the nylon membrane was hybridized overnight with a gene-specific probe.

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USES OF LM PCR

• Ligation-Mediated Polymerase Chain Reaction (LMPCR) is the most sensitive sequencing technique available to map single-stranded DNA breaks at the nucleotide level of resolution using genomic DNA. LMPCR has been adapted to map DNA damage and reveal DNA–protein interactions inside living cells.•However, the sequence context (GC content), the global break frequency and the current combination of DNA polymerases used in LMPCR affect the quality of the results.

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MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION (MLPA)

•MLPA is used to establish the copy number of up to 45 nucleic acid sequences in one single multiplex reaction. The method can be used for genomic DNA (including both copy number detection and methylation quantification) as well as for mRNA profiling, it permits multiple targets to be amplified with only a single primer pair, thus avoiding the resolution limitations of multiplex PCR.• The principle of MLPA is based on the identification of target sequences by hybridization of pairs of MLPA probes that bind to adjacent sequences and can then be 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 of interest.

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• 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 each MLPA probe has a unique length.•One PCR primer is fluorescently or isotopically labelled so that the MLPA reaction products 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 copy number of that target sequence.•Comparison of the electrophoresis profile of the tested sample to that obtained with a control sample enables the detection of deletions or duplications of genomic regions of interest

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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.

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TYPES OF MULTIPLEX PCR

1. 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 template

2. 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.

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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.• 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. •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.

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• 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. • 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.

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Advantages of Multiplex PCR

1. 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.

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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.

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USES OF MULTIPLEX PCR

• 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.

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METHYLATION-SPECIFIC PCR (MSP)

•Methylation-specific PCR (MSP) is used to identify patterns of DNA methylation at cytosine-guanine (CpG) islands in genomic DNA .• Target DNA is first treated with sodium bisulphite, which converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers. • Two amplifications are then carried out on the bisulphite-treated DNA: One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with uracil (corresponding to unmethylated cytosine). •MSP used in Q-PCR provides quantitative information about the methylation state of a given CpG island.

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• Treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. • Thus, bisulphite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA. • The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulphite conversion.

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• The MethyLight method is based on MSP, but provides a quantitative analysis using real-time PCR. •Methylated-specific primers are used, and a methylated-specific fluorescence reporter probe is also used that anneals to the amplified region. • In alternative fashion, the primers or probe can be designed without methylation specificity if discrimination is needed between the CpG pairs within the involved sequences. •Quantitation is made in reference to a methylated reference DNA. A modification to this protocol to increase the specificity of the PCR for successfully bisulphite-converted DNA (ConLight-MSP) uses an additional probe to bisulphite-unconverted DNA to quantify this non-specific amplification.

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Methylation-specific PCR flow

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NESTED PCR

• This PCR increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA.• Two sets (instead of one pair) of primers 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, may still consist of non-specifically amplified DNA fragments.

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• The product(s) are then used in a second PCR after the reaction is diluted with a set of second set “nested or internal” primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction.• The specificity of PCR is determined by the specificity of the PCR primers.• For example, if your primers bind to more than one locus (e.g. paralog or common domain), then more than one segment of DNA will be amplified. To control for these possibilities, investigators often employ nested primers to ensure specificity.

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•Nested PCR means that two pairs of PCR primers were used for a single locus. • The first pair amplified the locus as seen in any PCR experiment. • The second pair of primers (nested primers) bind within the first PCR product and produce a second PCR product that will be shorter than the first one. • The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers.

04/09/2023 46 NESTED PCR SCHEME

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. Nested PCR strategy. Segment of DNA with dots representing nondiscript DNA sequence of unspecified length. The double lines represent a large distance between the portion of DNA illustrated in this figure. The portions of DNA shown with four bases in a row represent PCR primer binding sites, though real primers would be longer.

The first pair of PCR primers (blue with arrows) bind to the outer pair of primer binding sites and amplify all the DNA in between these two sites

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PCR product after the first round of amplificaiton. Notice that the bases outside the PCR primer pair are not present in the product.

Second pair of nested primers (red with arrows) bind to the first PCR product. The binding sites for the second pair of primers are a few bases "internal" to the first primer binding sites.

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Final PCR product after second round of PCR. The length of the product is defined by the location of the internal primer binding sites.

USES OF NESTED PCR

When a complete genome sequence is known, it is easier to be sure you will not amplify the wrong locus but since very few of the world's genomes have been sequenced completely, nested primers will continue to be an important control for many experiments.

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QUANTITATIVE PCR (Q-PCR)•Used to measure the quantity of a PCR product (commonly in real-time).• It quantitatively measures starting amounts of DNA, cDNA or RNA. •Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. •Quantitative real-time PCR has a very high degree of precision. •QRT-PCR methods use fluorescent dyes, such as Sybr Green, EvaGreen or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time. • It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since RT-PCR commonly refers to reverse transcription PCR , often used in conjunction with Q-PCR.

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SY BR Green I

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ssD N A -- unbound dyem inimal fluorescence

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dsD N A -- bound dye >100fold inc rease fluorescence

TaqM an -- Hydrolysis P robe

M onitor acceptor fluorescence

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H ybrid ization probes

FRE T

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donor acceptor

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fluor quencher

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E xtension continues

Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe fluorogenic detection approaches for real-time PCR.

Two labeled probes must bind within the amplicon to generate a fluorescence signal as energy istransferred from the donor fluorophore to the acceptor fluorophore, a process known asfluorescence resonance energy transfer (FRET). These assays can be highly specific since twoprimers and two probes must bind. In addition, detection via hybridization probes is not dependenton a hydrolysis reaction, and thus, a melt analysis can be conducted. These assays can also bemultiplexed.

SYBR Green I is a fluorescent dye that upon intercalation into double stranded DNA exhibits anincrease in fluorescence intensity of greater than 100-fold. Advantages include the relatively lowcost of the dye, the fact that it will work with any primer set since it is not sequence specific, andthe ability to perform a melt analysis. The main disadvantage is that any nonspecific productsformed in the PCR, including primer dimers are also detected.

A TaqMan probe contains a fluorophore in close proximity to a quencher, such that the presence ofthe quencher blocks fluorescence. When bound to the target sequence, the probe is cleaved by thepolymerase. The necessity of a probe to bind internal to the amplicon results in increasedspecificity. These assays can be multiplexed, but have the disadvantages of difficult design andcost of the dual labeled probe and the inability to perform a melt analysis.

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REVERSE TRANSCRIPTION PCR (RT-PCR)

•A PCR designed for amplifying DNA from RNA. •Reverse transcriptase reverse transcribes RNA into cDNA, which is then amplified by PCR. •RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites. • If the genomic DNA sequence of a gene is known, RT-PCR can be used to map the location of exons and introns in the gene. • The 5' end of a gene (corresponding to the transcription start site) is typically identified by RACE-PCR (Rapid Amplification of cDNA Ends)

04/09/2023 54Reverse Transcription PCR flow

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With PCR, it is now possible to amplify and examine minute quantities of rare genetic material: the limit of this exploration being the single cell.

Single cell PCR has applications in many areas, and has great application especially in the field of prenatal diagnostics.

In prenatal diagnosis, single cell PCR has made possible preimplantation genetic analysis and the use of fetal cells enriched from the blood of pregnant women for the assessment of single-gene Mendelian disorders.

Single-cell PCR has not only proven its usefulness in diagnostics, but also lately has been very useful to basic scientists investigating immunological, neurological and developmental problems.

Single Cell PCR

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THERMAL ASYMMETRIC INTERLACED PCR (TAIL-PCR)

• This reaction is applied in the isolation of an unknown sequence flanking a known sequence. Within the known sequence, TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence . •Uses: TAIL-PCR as a powerful tool for amplifying insert end segments from P1, BAC and YAC clones, the amplified products were highly specific and suitable as probes for library screening and as templates for direct sequencing while the recover insert ends can also be used for chromosome walking and mapping.

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•Nested, insertion-specific primers are used together with arbitrary degenerate primers (AD primers), which are designed to differ in their annealing temperatures. •Alternating cycles of high and low annealing temperature yield specific products bordered by an insertion-specific primer on one side and an AD primer on the other. • Further specificity is obtained through subsequent rounds of TAIL-PCR, using nested insertion-specific primers. • The increasing availability of whole genome sequences renders TAIL-PCR an attractive tool to easily identify insertion sites in large genome tagging populations through the direct sequencing of TAIL-PCR products. • For large-scale functional genomics approaches, it is desirable to obtain flanking sequences for each individual in the population in a fast and cost-effective manner.

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OTHER TYPES OF PCR

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•Alu PCRThe pcr is performed using Alu primers designed to have recognition sequence of Alu restriction enzyme. Used as a method of obtaining a fingerprint of bands from an uncharacterized human DNA.

• LONG PCRLong 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.

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• AP-PCR

Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD) are methods of creating genomic fingerprints from species of which little is known about target sequence to be amplified.• COLONY PCRThe screening of bacterial (E.Coli) or yeast clones for correct ligation or plasmid products . 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 pre-inserted into autoclaved water. PCR is then conducted to determine if the colony contains the DNA fragment or plasmid of interest.

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TAP-PCR

AP PCR run at three different annealing temperature

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LAMP assay: (Loop-mediated isothermal amplification) • It is a Modified type of the PCR using 3-6 primers sets one of them is loop like primer. • This test use Bst-polymerase enzyme

(Bacillus stearothermophilus DNA Polymerase). • Using only two temperatures (63°C and 85°C for one hour), may be carry out in water bath.

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THE DIGITAL PCR• The Digital polymerase chain reaction simultaneously amplifies

thousands of samples, each in a separate droplet within an emulsion .

OVERLAP-EXTENSION 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 .

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 solid-support surface), conventional Solid Phase PCR (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)

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•Box PCR Box elements are repetitive sequence elements in bacterial genome such as Streptococcus genome. Single primer targeting to the repeats can be used to fingerprint bacterial species.•Competitive PCR(cPCR) this is a method used for quantifying DNA using real time PCR. A competitor internal standard is co amplified with the target DNA and the target is quantified from the melting curves of the target and its competitor.•Consensus PCR This PCR is carried out by using flanking primers to amplify repeat regions from a no. of species. In this case degenerate/consensus primers can be used for amplifying the flanking sequences.

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Degenerate PCR•In this instead of using specific PCR primers for a given sequence, mixed PCR primers will be used. •That is “wobbles” are inserted into the primers in case if the exact sequence of gene is not known, so that there will be more than one possibility for exact amplifications. •Degenerate PCR has proven to be a powerful tool to find ‘new’ gene or gene families. By aligning the sequences from a no. of related proteins the conserved and variables part can be determined. •Based on this information one can use conserved protein motifs for starting points for designing degenerate PCR primers. Degenerate oligonucleotide-primed PCR(DOR PCR)• PCR amplification of limited sample by using degenerate PCR primers is called DOR PCR.

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•Differential display PCR(DD PCR) It is used for cloning purpose; it combines the comparative analysis of several samples with the sensitivity of PCR.•Forensic PCR The VNTR locus is PCR amplified to compare DNA samples from different sources.•Hairpin PCR A method for error free DNA amplification for mutation detection. It first converts a DNA sequence to a hairpin. True mutations will maintain hairpin structure during amplification while PCR errors will disrupt the hairpin structure.•PCR ELISA The PCR products are labeled(digoxigenin) during amplification. A capture probe specific to PCR amplicon is used to immobilize the amplicon to immune-well plate. ELISA is then used against the label(anti-digoxigenin) to quantitate PCR products.

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TOUCHDOWN PCR (STEP-DOWN 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 (3-5°C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5°C) below the primer Tm. 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 .

MINIPRIMER PCR• This reaction uses a thermostable polymerase (S-Tbr) 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.

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• RACE PCR Rapid Amplification of cDNA Ends PCR. This technique is used to obtain 3’end of a cDNA; it requires some sequence information internal to mRNA under study. The sequence information obtained from this technique can be utilized to obtain full length cDNA clones using the 5’RACE technique.• Rep-PCR Is used for Genomic Fingerprinting of plant-associated bacteria and computer-assisted plant analyses. The genomic fingerprinting method employed is based on the use of DNA primers corresponding to naturally occurring interspersed repetitive elements in bacteria such as REP,ERIC and BOX elements.• Vectorette-PCR This method enables the amplification of specific DNA fragments in situation where sequence of only one primer is known. Thus it extends the application of PCR to the stretches of DNA where the sequence information is only available at one end.

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UNIVERSAL FAST WALKING PCR

• Used for genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific 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 (lariat-dependent nested PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE LaNe .

  VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR• 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 .

INTERSEQUENCE-SPECIFIC PCR (OR ISSR-PCR)• 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 Alu-PCR, and can help amplify sequences adjacent (or between) these repeats.

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REFERENCES

• Introduction to Plant Biotechnology By- H.S. Chawala

• Yasumasa Kimura et al. Optimization of turn-back primers in isothermal amplification (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089485/

• R. Manojkumar and Mrudula Varanat(2006) Polymerase Chain Reaction: Types and Its Application in theField of Biology. International journal of tropical medicine 1 (4):156-161

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