PCR: Introduction, Types, working mechanism, requirements to run, factor affecting , common source of errors and its application
Introduction of PCR
PCR stands for polymerase chain reaction and it is a fast and inexpensive technique used to “amplify” – copy – small segments of DNA or RNA . By this method, a single copy of a nucleic acid that is often difficult to detect by standard hybridization methods, is multiplied to ≥10^7 copies in a short period. In molecular biology, PCR revolutionized the study of DNA to such an extent that its creator, Kary B. Mullis, was awarded the Nobel Prize for Chemistry in 1993 from the National Human Genome Research Institute.
Types of PCR
Arbitrary primed PCR
Repeatative element PCR
Long Accurate PCR
Allele specific PCR
In Situ PCR
Real time Polymerase Chain Reaction
First significant increase in the amount of PCR product correlates to the initial amount of target template. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed. A significant increase in fluorescence above the baseline value measured during the 3-15 cycles indicates the detection of accumulated PCR product. Usually the protocol followed is depicted.
Real time PCR or quantitative PCR is a variation of the standard PCR technique used to quantify DNA or messenger RNA (mRNA) in a sample. Using sequence specific primers, the relative number of copies of a particular DNA or RNA sequence can be determined. We use the term relative since this technique tends to be used to compare relative copy numbers between tissues, organisms, or different genes relative to a specific housekeeping gene. The quantification arises by measuring the amount of amplified product at each stage during the PCR cycle. DNA/RNA from genes with higher copy numbers will appear after fewer melting, annealment, extension PCR cycles. Quantification of amplified product is obtained using fluorescent probes and specialized machines that measure fluorescence while performing temperature changes needed for the PCR cycles.
The starting template for a PCR reaction can be DNA or RNA. DNA is usually the appropriate template for studying the genome of the cell or tissue (as in inherited genetic diseases, somatic mutation in a tumor, or somatic rearrangement in lymphocytes) and for the detection of DNA viruses. For information on gene expression in a cell or tissue, or the presence of genomic RNA in a retrovirus such as HIV, RNA is the appropriate template. RNA can be better than genomic DNA for detecting structural changes in long genes, since amplifying the spliced RNA transcript instead of the genomic sequence greatly reduces the length of DNA to be handled without losing any of the coding regions where clinically significant deletions may be expected. RT-PCR combines cDNA synthesis from RNA templates with PCR to provide a rapid, sensitive method for analyzing gene expression (Figure 3). RT-PCR is used to detect or quantify the expression of mRNA, often from a small concentration of target RNA.
The template for RT-PCR can be total RNA or poly (A)+ selected RNA. RT reactions can be primed with random primers, oligo(dT), or a gene-specific primer (GSP) using a reverse transcriptase. RT-PCR can be carried out either in two-step or one- step formats. In two-step RT-PCR, each step is performed under optimal conditions. cDNA synthesis is performed first in RT buffer and one tenth of the reaction is removed for PCR5. In one-step RT-PCR, reverse transcription and PCR take place sequentially in a single tube under conditions optimized for both RT and PCR.
Multiplex Polymerase Chain Reaction
In multiplex PCR, two or more unique target sequences can be amplified simultaneously. It can be used in diagnostic assays that use one set of primers to amplify an internal control to verify the integrity of the PCR while the second set of primer is targeted to DNA sequence of interest. Absence of control amplicon indicates that PCR conditions were not met and the PCR may have to be repeated. Multiplex PCR can also be used to test for different organism on a single specimen. The primers are so designed that each amplification product is a unique size allowing detection and identification of specific organisms. Primer sets should have similar annealing temperatures. A 10°C difference in annealing temperature between primer sets can lead to widely different amount of amplified products or no detectable amplification of one target or other. A limitation of multiplex PCR is that mixing different primers can cause some interference in the amplification process and the optimization of conditions becomes difficult as the number of primer pairs increase. Multiplex PCRs with targets that differ widely in size often favors amplification of shorter targets over larger ones resulting in different amounts of amplified products.
Nested Polymerase Chain Reaction
Nested PCR uses two sets of amplification primers. The first set of primers is used to amplify a target sequence and the second set of primers are used to amplify a region within the first target sequence. Essentially, this involves amplification of a sequence internal to an amplicon. Because the production of second amplicon depends on the successful production of the first amplicon, production of second amplicon automatically validates the accuracy of the first amplicon. Nested PCR may be performed in a single tube method or two-tubes method. In the single tube method, both the external and internal primer sets are added at the same time. There are two ways to accomplish nested PCR in single tube, one method involving physical separation of two sets of reaction and the other method involving difference in annealing temperature of primers. In physical separation method, the tube is filled with primary mixture consisting of target DNA, first (external) set of primers and other necessary components. This is overlaid with thick mineral oil layer into which second (external) set of primers and other components have been inserted. After the first round of 25-30 cycles of PCR, the reaction mixture is spun to mix the external primer and other components held inside the oil overlay. A second round of 25-30 cycles are performed and products are analysed. In the differential annealing temperature method, the reaction mixture is set up to contain both the outer and inner sets of primer. These primer sets are so designed that outer primer pair hybridizes with the target at a temperature lower than what is required for hybridization of inner primer sets. Switching to
higher temperature allows amplification of the internal product. In the two tube procedure, target is amplified using only the outer primer sets after 25-30 PCR cycles. After this, the tube is opened and the mixture is transferred to another tube containing inner primer sets which hybridizes to the amplicon generated using outer primes. After running 25-30 cycles, the products are analyzed by standard methods. Nested PCR makes the reaction very specific and alleviates false positive reactions that may occur with other PCR systems.
Competitive PCR is a quantitative PCR, which is used to quantify the amount of nucleic acid (DNA or RNA) in the sample. The method involves a competition between the target nucleic acid and the competitive DNA for amplification process. The competitive DNA requires the same primer pair and is added in known concentration. It differs from the target DNA in size or presence of unique restriction site. The internal DNA standard is an identical sequence to an unknown DNA sample being analyzed except that it contains either a deletion or insertion. This difference is required to accurately differentiate target DNA from competitor DNA after the amplification process. Once amplification of target DNA is performed along with DNA competitor, competitive PCR occurs due to the competition for the use of the primers. Because of the competition, the ratio of the amount between two amplified products reflects the ratio between the target DNA and DNA competitor. So, the amount of the target DNA can be estimated by comparing with the concentration of DNA competitor. Target DNA or RNA can be relatively quantified compared with the initial amount of competitor.
Resulting amplicons of fractional log-dilutions of the internal standard are matched with those of target on separation by agarose gel electrophoresis, which allows an estimation of the amount of transcripts in the unknown sample. Initial amount of target DNA or RNA can be estimated by T/C ratio, where T is the amount of amplified product from target DNA or RNA and C is the amount of amplified product from competitor. Initial amount of target DNA or RNA will correspond to the competitor’s amount, when T/C
Amplified Fragment Length Polymorphism Polymerase Chain Reaction
Amplified Fragment Length Polymorphism polymerase chain reaction, also called AFLP PCR was originally described by Zabeau et al., 1993. The key feature of AFLP–PCR is its capacity for the simultaneous screening of many different DNA regions distributed randomly throughout the genome. This technique combines the strengths of two methods, the replicability of restriction fragment analysis and the power of the PCR. AFLP is composed of 3 steps:
1) Cellular DNA is digested with one or more restriction enzymes. Typically this involves a combination of two restriction enzymes; a restriction enzyme that cuts frequently (MseI, 4 bp recognition sequence) and one that cuts less frequently (EcoRI, 6 bp recognition sequence).
2) AFLP adaptors are joined (ligated) to these ends
3) Pre-selective PCR is performed using primers which match the linkers and restriction site specific sequences. A pre-selective PCR amplification is done using primers complementary to each of the two adaptor sequences, except for the presence of one additional base at the 3′ end.
4) Electrophoretic separation and amplicons on a gel matrix, followed by visualization of the band pattern. Adaptor ligations are performed in the presence of restriction enzymes such that any fragment-to- fragment ligations are immediately recleaved by the restriction enzyme. The adaptor is designed so that ligation of a fragment to an adaptor does not reconstitute the restriction site. The end sequences of each fragment now consist of the adaptor sequence and the remaining part of the restriction sequence, which serve as priming sites in the subsequent AFLP–PCR. Depending on genome size, restriction-ligation generates thousands of adapted fragments. To achieve selective amplification of a subset of these fragments, primers are extended into the unknown part of the fragments, usually one to three arbitrarily chosen bases beyond the restriction site. To minimize artifacts, most protocols incorporate two amplifications. The first is performed with a single-bp extension, followed by a more selective primer with up to a 3-bp extension. In a second, “selective”, PCR, using the products of the first as template, primers containing two further additional bases, chosen by the user, are used. Because of the high selectivity, primers differing by only a single base in the AFLP extension amplify a different subset of fragments. By using combinations of primers with different extensions, a series of AFLP amplifications can thus screen a representative fraction of the genome. AFLP–PCR products can be separated and scored with a variety of techniques, ranging from simple agarose gel electrophoresis to automated genotyping. Polyacrylamide gel electrophoresis provides maximum resolution of AFLP banding patterns to the level of single-nucleotide length differences.
Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction -based markers for the rapid screening of genetic diversity. AFLP methods rapidly generate hundreds of highly replicable markers from DNA of any organism; thus, they allow high-resolution genotyping of fingerprinting quality. AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA. AFLP can be also used for genotyping individuals for a large number of loci using a minimal number of PCR reactions. AFLP markers have emerged as a major new type of genetic marker with broad application in systematics, pathotyping, population genetics, DNA fingerprinting and quantitative trait loci (QTL) mapping.
Asymmetrical Polymerase Chain Reaction
Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to 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 (Melting temperature|Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. Asymmetric PCR potentially circumvents the problem of amplicon strand reannealing by using unequal primer concentrations. Depletion of the limiting primer during the exponential amplification
results in the linear synthesis of the strand extended from the excess primer.
Asymmetric Polymerase Chain Reaction also requires extensive optimization to identify the proper primer ratios, the amounts of starting material, and the number of amplification cycles that can generate reasonable amounts of product for individual template target combinations.
Assembly Polymerase Chain Reaction
Assembly PCR is the artificial synthesis of long DNA sequences by performing Polymerase Chain Reaction 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.
This technique 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.
This is a technique that reduces non-specific amplification during the initial set up stages of the polymerase chain reaction. The technique 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 Polymerase Chain Reactionis achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.
A method used to allow polymerase chain reaction when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.
This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking and DNA footprinting.
Used to amplify DNA over the entire length up to 25 kb of genomic DNA segments cloned.
Arbitrarily primed polymerase chain reaction
The arbitrarily primed polymerase chain reaction (AP-PCR) is a PCR-based DNA fingerprinting technique using primers whose nucleotide sequence is arbitrarily chosen (Welsh and McClelland 1990; Williams et al. 1990). This method has also been called random amplified polymorphic DNA (RAPD).
Colony polymerase chain reaction- the screening of bacterial (E. coli) or yeast clones for correct ligation or plasmid products. Pick a bacterial colony with an autoclaved toothpick, swirl it into 25 μl of TE autoclaved dH2O in an microfuge tube. Heat the mix in a boiling water bath (90-100°C) for 2 minutes. Spin sample for 2 minutes high speed in centrifuge. Transfer 20 μl of the supernatant into a new microfuge tube.Take 1-2 μl of the supernatant as template in a 25 μl PCR standard. PCR reaction.
Repetitive element PCR
Repetitive element polymerase chain reaction (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species.
Long accurate PCR
Extended or longer than standard polymerase chain reaction, meaning over 5 kilobases (frequently over 10 kb). Long PCR is useful only if it is accurate. Thus, special mixtures of proficient polymerases along with accurate polymerases such as Pfu are often mixed together and its application- to clone large genes.
Allele-specific polymerase chain reaction
Allele-specific polymerase chain reaction (ASPCR) is an application of the polymerase chain reaction that permits the direct detection of any point mutation in human DNA by analyzing the polymerase chain reaction products in an ethidium bromide-stained agarose or polyacrylamide gel. ASPCR works because an oligonucleotide primer that forms a 3′ mismatch with the DNA template will be refractory to primer extension by Thermus aquaticus DNA polymerase
In Situ PCR
In situ polymerase chain reaction (IS-PCR) describes the primer-driven amplification of a DNA or RNA template by polymerase chain reaction and its subsequent detection within the confines of a histological tissue section.
The process of amplifying the target begins with designing a pair of primers complementary to the two regions spanning the target on either strand. The two strands of the target DNA are separated (denaturation) by heating and the primers are applied. The primers bind to their targets on either strands.These primers are then elongated by DNA polymerase enzyme complimentary to the nucleotides on the target on both strands. Thus, each single strand of the target gets its complimentary strand, resulting in production of two target DNA molecules from a single DNA molecule. This process is repeated several times in order to obtain larger number of copies. The amplified target DNA segments are called amplicons. All the steps of polymerase chain reaction are performed on the reaction mixture consisting of target DNA, primer pairs, thermostable DNA polymerase, deoxynucleotides (dATP, dTTP, dGTP & dCTP), buffer and Mg salt in the same test tube. Primers are short, single stranded oligonucleotide DNA that are 20-30 nucleotides that are chemically synthesized. Primes are always designed in pairs, complementary to opposite strands of the target. Primers are so designed that they hybridize with the target 50-3000 nucleotides apart from each other. In other words, primers bind to the region flanking target sequences that are 50-3000 nucleotides long. For the primers to bind, the target nucleic acid is denatured by heating at 90-100°C. The primers are added and cooled to 40-65° C to allow the primers to hybridize. DNA polymerase is added to elongate the primers complementary to the target. These enzymes need a portion of complementary sequence to start the elongation and the hybridized primers serve this function. Depending on the nature of polymerase enzyme, the temperature may be raised to allow polymerase enzyme to synthesize complementary strands. The newly formed dsDNA is melted and the process is repeated for several cycles. Commonly used set of cycling temperature included denaturation of dsDNA at 94°C for 15 seconds to 1 minute; hybridization of oligonucleotide primers at 52°C for 15 seconds to 2 minutes; extension of primers by DNA polymerase (Taq) at 72° C for 15 seconds to 3 minutes. The three steps are repeated for 25-35 cycles. In order to overcome the problem of adding the polymerase enzyme after each cycle as they are destroyed at high temperatures, thermostable polymerase enzymes such as Taq is used. Taq DNA polymerase enzyme is obtained from Thermus aquaticus, a bacterium that lives in hot water springs. Once added, it is adequate for 30 or more cycles. Other thermostable polymerases are Pfu, Pwo and Tth. The process of transferring the tubes to different temperatures has been circumvented by the use of automated machines called thermocyclers. polymerase chain reaction is an exponential amplification system such that after n number of cycles, there is (1+x)n times as much target as was present initially; where x is the mean efficiency of the reaction for each cycle. After a standard run of 30 cycles over a period of 2-3 hours, theoretically the original target molecule is amplified 230 times (10^9 fold). However, polymerase chain reaction is usually less efficient than theoretical and the actual practical amplifications are only 10^6-10^7 folds, which is due to various factors such as inactivation of Taq DNA polymerase, shortage of nucleotide substrates, shortage of primer, inhibition by pyrophosphate and re-annealing of amplified DNAs. After certain number of polymerase chain reaction cycles, it attains plateau phase. The plateau phase of it indicates that almost same amount of amplified products will be obtained, regardless of the initial amount of the templates, by sufficient cycles of polymerase chain reaction . For targets up to 1000 bp, 20-30 cycles of PCR are performed, but if organisms are present in low numbers 45 cycles may be required. Even though the system has the ability to detect one copy of DNA in the sample, detection is dependent on the ability of the primers to locate and anneal to the target copy and optimum polymerase chain reaction conditions.
Requirements for test
PCR reaction mixture that contains-
Nuclease free water
where as master mix contains-
PCR tube with cap
Micro pipettes with filter tips
Refrigerator and deep fridge
Factors affecting PCR
Concentration of Mg2+; since thermostable polymerase are Mg dependent enzymes. Each combination of target DNA and primer require unique concentration of Mg2+.
Concentration and source of thermostable polymerase
Concentration and purity of both target DNA and primer
Denaturing temperature, annealing temperature and time
Number of cycles
Common problems in polymerase chain reaction are false negatives due to presence of polymerase chain reaction inhibitors, poor nucleic acid isolation and poor amplification efficiency and false positives due to contaminations.
Common sources of error are
False positive reactions are caused by contamination with a new or previously amplified DNA
Non-specific primer hybridization. Binding of primers to area other than desired region resulting in false positive results.
Primer dimer formation. This condition can arise if the two primers used are complimentary to each other and end up hybridizing with each other instead of hybridizing with the target. This may lead to little or no amplification of target sequence.
Background hybridization occurs as a result of non-specific binding of only one of the primers.This results in the amplification of the target DNA thirty times after 30 cycles and not exponentially.
Primer artifact formation can occur in conditions of low stringency, small target amounts, too much enzyme in early cycles, high primer concentration and excessive thermal cycling. This often occurs when the polymerase enzyme generates new primer binding sites for the same or other
primer. This decreases the efficiency of polymerase chain reaction by consuming primer and competing with target for other reaction components.