POLYMERASE CHAIN REACTION (PCR): Principle, features, components used, enzyme sourse & importance, hot start technique, key notes to follow the procedure, pictures of PCR including agarose gell pattern.

Introduction

PCR Definition

The polymerase chain reaction (PCR) is a primer-mediated enzymatic amplification of specifically cloned or genomic DNA sequences. This PCR process, invented more than a decade ago, has been automated for routine use in laboratories worldwide. The template DNA contains the target sequence, which may be tens or tens of thousands of nucleotides in length. A thermostable DNA polymerase such as Taq DNA polymerse catalyzes the buffered reaction in which an excess of an oligonucleotide primer pair and four deoxynucleoside triphosphates (dNTPs) are used to make millions of copies of the target sequence. Although the purpose of the PCR process is to amplify template DNA, a reverse transcription step allows the starting point to be RNA.

Scope of PCR Applications

PCR is widely used in molecular biology and genetic disease studies to identify new genes. Viral targets, such as HIV-1 and HCV, can be identified and quantified by PCR. Active gene products can be accurately quantitated using RNA-PCR. In such fields as anthropology and evolution, sequences of degraded ancient DNAs can be tracked after PCR amplification. With its exquisite sensitivity and high selectivity, PCR has been used in wartime human identification and validation in crime labs for mixed-sample forensic casework. In the realm of plant and animal breeding, PCR techniques are used to screen for traits and to evaluate living four-cell embryos. Environmental and food pathogens can be quickly identified and quantitated at high sensitivity in complex matrices with simple sample preparation techniques.

PCR Process

The PCR process requires a repetitive series of the three fundamental steps that defines one PCR cycle: double-stranded DNA template denaturation, annealing of two oligonucleotide primers to the single-stranded template, and enzymatic extension of the primers to produce copies that can serve as templates in subsequent cycles. The target copies are double-stranded and bounded by annealing sites of the incorporated primers. The 3' end of the primer should complement the target exactly, but the 5' end can actually be a non-complementary tail with restriction enzyme and promotor sites that will also be incorporated. As the cycles proceed, both the original template and the amplified targets serve as substrates for the denaturation, primer annealing, and primer extension processes. Since every cycle theoretically doubles the amount of target copies, a geometric amplification occurs. Given an efficiency factor for each cycle, the amount of amplified target Y produced from an input copy number X after n cycles is;

Y = X (1 = efficiency)ⁿ

With this amplification power, 25 cycles could produce 33 million copies. Every extra 10 cycles produces 1024 more copies. Unfortunately, the process becomes self-limiting and amplification factors are generally between 105- and 109-fold. Excess primers and dNTPs help drive the reaction that commonly occurs in 10 mM Tris-HCl buffer, pH 8.3 (at room temperature). In addition, 50 mM KCl is present to provide proper ionic strength and magnesium ion is required as an enzyme cofactor. The denaturation step occurs rapidly at 94–96°C. Primer annealing depends on the Tm, or melting temperature, of the primer: template hybrids. Generally, one uses a predictive software program to compute the Tms based on the primer’s sequence, their matched concentrations, and the overall salt concentration. The best annealing temperature is determined by optimization. Extension occurs at 72°C for most templates. PCR can also easily occur with a two-temperature cycle consisting of denaturation and annealing/extension.

Carryover Prevention

PCR has the potential sensitivity to amplify single molecules, so PCR products that can serve as templates for subsequent reactions must be kept isolated after amplification. Even tiny aerosols can contain thousands of copies of carried-over target molecules that can convert a true negative into a false positive. In general, dedicated pipetors, pipet tips with filters, and separate work areas should be considered and/or designated for RNA or DNA sample preparation, reaction mixture assemblage, the PCR process, and the reaction product analysis. As with any high sensitivity technique, the judicious and frequent use of positive and negative controls is required for each amplification. Through the use of dUTP instead of dTTP for all PCR samples, it is possible to design an internal biochemical mechanism to attack the PCR carryover problem. These PCR products are dU-containing and can be cloned, sequenced, and analyzed as usual. Pretreatment of each PCR reaction with uracil-N glycosylase (UNG), which catalyzes the removal of uracil from single- and double stranded DNA, will destroy any PCR product carried over from previous reactions, leaving the native T-containing sample ready for amplification.

Hot Start

PCR is conceptualized as a process that begins when thermal cycling ensues. The annealing temperature sets the specificity of the reaction, assuring that the primary primer binding events are the ones specific for the target in question. In preparing PCR amplification on ice or at room temperature, however, the reactants are all present for nonspecific primer annealing to any single-stranded DNA present. Because DNA polymerases have some residual activity even at lower temperatures, it is possible to extend these misprimed hybrids and begin the PCR process at the wrong sites. To prevent this mispriming/misextension, a number of “Hot Start” strategies have been developed. In Hot Start PCR, a key reaction component essential for polymerase activity is withheld or separated from the reaction mixture until an elevated temperature is reached. To separate an essential component from the reaction mixture in order to delay amplification, the following techniques can be utilized:

Manual Hot Start

In Manual Hot Start, a key reaction component such as Taq DNA polymerase or MgCl2 is withheld from the original amplification mixture and added to the reaction when the temperature within the tube exceeds the optimal annealing temperature, i.e., above 65°–70°C.

Physical Barrier Hot Start

In AmpliWax PCR gem-facilitated Hot Start, reaction components are divided into two mixes, and separated by a solid wax layer within the reaction tube. During the initial denaturation step, the wax layer melts at 75°–80°C allowing the two reaction mixes to combine through thermal convection.

Monoclonol Antibodies to DNA Polymerases Hot Start

In polymerase-antibody Hot Start, a PCR preincubation step is added, during which a heat-sensitive antibody attaches to the DNA polymerase [Taq or recombinant Thermus thermophilus (rTth)] inactiving the enzyme within the reaction mixture. As the temperature within the tubes rises, the antibody detaches and is inactivated, setting the polymerase free to begin polymerization. 1.5.4. Modified DNA Polymerases for Hot Start, i.e., AmpliTaq Gold from Applied Biosytems With AmpliTaq Gold, Hot Start is achieved with a chemically modified Taq DNA polymerase. The modification blocks the polymerase activity until it is reversed by a high temperature, pre-PCR incubation (e.g., 95°C for >10 min). The pre-PCR incubation links directly to the denaturation step of the first PCR cycle. So, the reaction mixture never sees active polymerase below the optimal primer annealing temperature. If the pre-PCR incubation is omitted, the modification is reversed during the PCR cycling, and polymerase activity increases slowly. In addition to a Hot Start, this provides a time release effect, where polymerase activity builds as the DNA substrate accumulates.

Oligonucleotide Inhibitors of DNA Polymerases for Hot Start

In polymerase-inhibitor Hot Start, DNA polymerase-binding oligonucleotides are added to the PCR amplification, keeping the enzyme inactive at ambient temperatures. Increasing the temperature dissociates the inhibitor from the enzyme, setting it free to begin polymerization. Moreover, inhibition is thermally reversible.

PCR Achievements PCR has been used to speed the human genome discovery and for early detection of viral diseases. Single sperm cells to measure crossover frequencies can be analyzed and four-cell cow embryos can be typed. Trace forensic evidence of even mixed samples can be analyzed. Single-copy amplification requires some care, but is feasible for both DNA and RNA. True needles in haystacks can be found simply by amplifying the needles. PCR facilitates cloning of DNA sequences and forms a natural basis for cycle sequencing by the Sanger method (17). In addition to generating large amounts of template for cycle sequencing, PCR has been used to map chromosomes and to analyze both large and small changes in chromosome structure.

PCR Enzymes

The choice of the DNA polymerase is determined by the aims of the experiment. There are a variety of commercially available enzymes to choose from that differ in their thermal stability, processivity, and fidelity as depicted in Table 1. The most commonly used and most extensively studied enzyme is Taq DNA polymerase, e.g., AmpliTaq DNA polymerase.

Primers

PCR Primers are short oligodeoxyribonucleotides, or oligomers, that are designed to complement the end sequences of the PCR target amplicon. These synthetic DNAs are usually 15–25 nucleotides long and have approximately 50–60% G + C content. Because each of the two PCR primers is complementary to a different individual strand of the target sequence duplex, the primer sequences are not related to each other. In fact, special care must be taken to assure that the primer sequences do not form duplex structures with each other or hairpin loops within themselves. The 3' end of the primer must match the target in order for polymerization to be efficient, and allele-specific PCR strategies take advantage of this fact. In screening for potential sequences and their homology, primer design software packages such as Oligo® (National Biosciences, Plymouth, NC) and online search sites such as BLAST (NCBI, www.ncbi.nlm.nih.gov/BLAST/), can be utilized. To screen for mutants, a primer complementary to the mutant sequence is used and results in PCR positives, whereas the same primer will be a mismatch for the wild type and does not amplify. The 5' end of the primer may have sequences that are not complementary to the target and that may contain restriction sites or promotor sites that are also incorporated into the PCR product. Primers with degenerate nucleotide positions every third base may be synthesized in order to allow for amplification of targets where only the amino acid sequence is known. In this case, early PCR cycles are performed with low, less stringent annealing temperatures, followed by later cycles with high, more stringent annealing temperatures. A PCR primer can also be a homopolymer, such as oligo (dT) 16, which is often used to prime the RNA PCR process. In a technique called RAPDS (randomly amplified polymorphic DNAs), single primers as short as decamers with random sequences are used to prime on both strands, producing a diverse array of PCR products that form a fingerprint of a genome (20). Often, logically designed primers are less successful in PCR than expected, and it is usually advisable to try optimization techniques for a practical period of time before trying new primers frequently designed near the original sites.

T m Predictions

DNA duplexes, such as primer-template complexes, have a stability that depends on the sequence of the duplex, the concentrations of the two components, and the salt concentration of the buffer. Heat can be used to disrupt this duplex. The temperature at which half the molecules are single-stranded and half are double-stranded is called the Tm of the complex. Because of the greater number of intermolecular hydrogen bonds, higher G+C content DNA has a higher Tm than lower G+C content DNA. Often, G + C content alone is used to predict the Tm of the DNA duplex, however, DNA duplexes with the same G + C content may have different Tm values. A simple, generic formula for calculating the Tm is: Tm = 4(G+C) + 2(A+T) °C. A variety of software packages are available to perform more accurate Tm predictions using sequence information (nearest neighbor analysis) and to assure optimal primer design, e.g., Oligo, BLAST, or Melt (Mt. Sinai School of Medicine, New York, NY).

Because the specificity of the PCR process depends on successful primer binding events at each amplicon end, the annealing temperature is selected based on the consensus of the melting temperatures (within 2– 4°C) of the two primers. Usually, the annealing temperature is chosen a few degrees below the consensus annealing temperatures of the primers. Different strategies are possible, but lower annealing temperatures should be tried first to assess the success of amplification to find the stringency required for best product specificity.

PCR Samples

Types

The PCR sample type may be single- or double-stranded DNA of any origin— animal, bacterial, plant, or viral. RNA molecules, including total RNA, poly (A+) RNA, viral RNA, tRNA, or rRNA, can serve as templates for amplification after conversion to so-called complementary DNA (cDNA) by the enzyme reverse transcriptase (either MuLV or recombinant, rTth DNA polymerase).

Amount

The amount of starting material required for PCR can be as little as a single molecule, compared to the millions of molecules needed for standard cloning or molecular biological analysis. As a basis, up to nanogram amounts of DNA cloned template, up to microgram amounts of genomic DNA, or up to 105 DNA target molecules are best for initial PCR testing.

Purity

Overall, the purity of the DNA sample to be subjected to PCR amplification need not be high. A single cell, a crude cell lysate, or even a small sample of degraded DNA template is usually adequate for successful amplification. The fundamental requirements of sample purity must be that the target contains at least one intact DNA strand encompassing the amplified region and that the impurities associated with the target be adequately dilute so as to not inhibit enzyme activity. However, for some amplifications, such as long PCR, it may be necessary to consider the quality and quantity of the DNA sample.

For example,

1. When more template molecules are available, there is less occurrences of false positives caused by either cross-contamination between samples or “carryover” contamination from previous PCR amplifications;

2. When the PCR amplifications lacks specificity or efficiency, or when the target sequences are limited, there is a greater chance of inadequate product yield; and

3. When the fraction of starting DNA available to PCR is uncertain, it is increasingly difficult to determine the target DNA content.

NOTE

Even though the PCR process has greatly enhanced scientific studies, a variety of problems with the process, easily revealed by ethidium-bromides-stained agarose gel electrophoresis, can and may need to be considered when encountered. For example, unexpected molecular weight size bands (nonspecific banding) or smears can be produced. These unexpected products accumulate from the enzymatic extension of primers that annealed to nonspecific target sites. Second, primer-dimer (approx 40–60 bp in length, the sum of the two primers) can be produced. Primer-dimer can arise during PCR amplification when the DNA template is left out of the reaction, too many amplification cycles are used, or the primers are designed with partial complementarity at their 3' ends. Note, an increase in primer-dimer formation will decrease the production of the desired product. Third, Taq DNA polymerase, which lacks the 3'-5' exonuclease “proofreading” activity, will occasionally incorporate the wrong base during PCR extension. The consequences of Taq misincorporations usually have little effect, but should be considered during PCR cloning and subsequent cycle sequencing.

PCR amplification for user-selected templates and primers are considered “failures” when 1) no product bands are observed; 2) the PCR product band is multibanded; or 3) the PCR product is smeared. These “failures” can be investigated and turned into successful PCR by manipulation of a number of variables, such as enzyme and salt concentrations, denaturation and anneal/extend times and temperatures, primer design, and hot start procedures. When no desired PCR product band is observed, initially verify the enzyme addition and/or concentration by titrating the enzyme concentration. Second, the magnesium ion concentration is also critical, so care should be taken not to lower the magnesium ion molarity on addition of reagents (i.e., buffers containing EDTA will chelate out the magnesium ion). The denaturation and anneal/extend times and temperatures may be too high or too low, causing failures, and can be varied to increase reaction specificity. Finally, the chemical integrity of the primers should be considered. In cases where the PCR product band is multibanded, consider raising the anneal temperature in increments of 2°C and/or review the primer design and composition. If a smear of the PCR product band is seen on an ethidium-bromide-stained agarose gel, consider the following options initially, individually, or in combination: decreasing the enzyme concentration, lowering the magnesium ion concentration, lengthening and/ or raising the denaturation time and temperature, shortening the extension time, reducing the overall cycle number, and decreasing the possibility of carryover contamination. Finally, in PCR amplifications where the PCR product band was initially observed, and on later trials a partial or complete loss of the product bands is observed, consider testing new aliquots of reagents and decreasing the possibility of carryover contamination. For PCR amplifications using a modified DNA polymerase such as AmpliTaq Gold, poor product amplification can occur owing to inadequate activation of the Hot Start polymerase. Incubation time, temperature, and pH are critical for Hot Start polymerase activation. Contaminants added with the target, whether remnants from the sample’s source or artifacts of the sample’s preparation, can affect the PCR pH. Contaminants may also directly inhibit the polymerase. Hot Start polymerase activation begins during the pre-PCR activation step and continues through the PCR cycles’ denaturation steps. The temperature and duration of these steps and the total number of PCR cycles should be optimized. Additional PCR cycles may increase specific product yield without increasing background in a Hot Start PCR. Raising the temperature above 95°C for any PCR step may irreversibly denature the polymerase.

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Saturday, 14 March 2015