PCR technology
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PCR (polymerase chain reaction) is a technique used to amplify a specific sequence of DNA. It involves cycling between heating and cooling steps to denature and copy the DNA. During each cycle, the amount of target DNA doubles, allowing millions of copies to be produced in a few hours. It uses primers that are complementary to the target sequence and a thermostable DNA polymerase to copy the target. The basic steps involve denaturing the DNA, annealing the primers, and extending the primers to copy the target. Nested PCR and other variations allow amplification of rare sequences or detection of gene expression.
PCR technology
- 1. 1 of 7 PCR Technology The polymerase chain reaction (PCR): copying (amplifying) specific sequences in DNA (or in RNA: reverse transcriptase) millions of copies are produced within a few hours. PCR is a technique analogous to the biosynthesis of DNA (replication) that takes place in living cells .(DNA amplification) PCR is used to amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence. These are extended towards each other by a thermostable DNA polymerase in a reaction cycle of three steps: denaturation, primer annealing and polymerization. Characteristics simple, fast, amplification of a target DNA, specificity and sensitivity General PCR Procedure: 1) Design specific or suitable primers by using manual method or software or from articles 2) Extraction and purification of DNA from sample 3) PCR mixture (mix template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer in PCR tubes 4) PCR cycle ( the mixture is cycled in thermal cycler many times (usually 30) through temperatures that permit denaturation, annealing, and synthesis to exponentially amplify a product of specific size and sequence 5) Analysis of PCR products (amplified DNA): The PCR products are then displayed on an appropriate gel (agarose , polyacrylamide) , for the PCR product size expected and examined for yield and specificity. Many important variables can influence the outcome of PCR. PCR reaction mixture (1) DNA templates (2) A pair of primers (3) DNA polymerase (Taq polymerase) (4) dNTPs (5) Buffer pH, salt, Mg 2+ The PCR cycle: Initial denaturation (strand separation) 940 C 3min 1 cycles Denaturation (strand separation) 940 C 1min 35 cycles Primer annealing (Primer binding) 550 C-630 C 1min Extension (DNA synthesis) 720 C 1min Final extension (DNA synthesis) 720 C 5min 1 cycles Storage 40 C Hold
- 2. 2 of 7 The basic steps in a PCR experiment are as follows 1. The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature. 2. The mixture is cooled down to 50–60°C. The two strands of each molecule could join back together at this temperature, but most do not because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions. Annealing It is critical that the primers anneal stably to the template, because, this can affect the specificity of the reaction. DNA–DNA hybridization is a temperature- dependent phenomenon. If the temperature is too high no hybridization takes place; instead the primers and templates remain dissociated. However, if the temperature is too low, mismatched hybrids—ones in which not all the correct base pairs have formed—are stable 3. The temperature is raised to 72°C. This is a good working temperature for the Taq DNA polymerase that is present in the mixture. The Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules, during this step of the PCR. 4. The temperature is increased back to 94°C. The double-stranded DNA molecules, each of which consists of one strand of the original molecule and one new strand of DNA, denature into single strands. This begins a second cycle of denaturation– annealing–synthesis, at the end of which there are eight DNA strands. By repeating the cycle 30 times the double-stranded molecule that we began with is converted into over 130 million new double-stranded molecules, each one a copy of the region of the starting molecule delineated by the annealing sites of the two primers. Figure ( ) the basic steps in the polymerase chain reaction
- 3. 3 of 7 Template: Almost any source that contains one or more intact target DNA molecule can, in theory, be amplified by PCR, This includes DNA prepared from blood, sperm or any other tissue, from older forensic specimens, from ancient biological samples or in the laboratory from bacterial colonies or phage plaques as well as purified DNA. Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed. Enzymes: Thermostable DNA polymerases which have been isolated and cloned from a number of thermophilic bacteria are used for PCR. ~1kb / min is the rate of DNA synthesis by this enzyme. The most common is Taq polymerase from Thermus aquaticus. It survives the denaturation step of 95°C for 1–2 min, having a half-life of more than 2 h at this temperature. Because it has no associated 3′ to 5′ proofreading exonuclease activity, Taq polymerase is known to introduce errors when it copies DNA – roughly one per 250 nt polymerized. For this reason, other thermostable DNA polymerases with greater accuracy are used for certain applications. Primers: A pair of oligonucleotides of about 18–30 nt with similar G+C content will serve as PCR primers as long as they direct DNA synthesis towards one another. The oligonucleotide primers are the most critical element in terms of successful PCR. If the primers are incorrectly designed, the experiment will fail, possibly because no amplification occurs, or possibly because the wrong fragment, or more than one fragment, is amplified. One consideration is distance between the primers. Smaller DNA fragments are amplified more efficiently than longer DNA fragments. The DNA fragment to be amplified should not be greater than about 3 kb in length and ideally less than 1 kb. The key to the PCR lies in the design of the primers: Typical PCR primers are anything between 18-28 nucleotides in length The G+C composition should ideally be similar to that of the desired amplicon and should in general be between 50-60% The calculated Tm for a primer pair should be balanced A Tm 55°C -72°C is desired (62-65°C is best) Check for complementarity in 3' ends of primer pairs - this lead to primer - dimer artifacts Avoid any significant secondary structure within primers i.e. internal palindromic sequences Runs of 3 or more C’s and G’s at 3' ends promote mispriming in G/C rich regions Palindromic sequences within the primers should be avoided Avoid an A and especially a T at the 3’ end of a primer (this allow ‘breathing’ in the hybridisation of the primer to the template) Avoid any potential mismatches in the 3’end of primers For short oligonucleotides (<25 nt), the annealing temperature (in °C) can be calculated using the formula: Tm = 2(A+T) + 4(G+C), where Tm is the melting temperature and the annealing temperature is approximately 3–5°C lower. If the DNA sequence being amplified is known, then primer design is relatively easy. The region to be amplified should be inspected for two suitable sequences of about 20 nt with a similar G+C content, either side of the region to be amplified (e.g. the site of mutation in certain cancers). If the PCR product is to be cloned, it is sensible to include the sequence of unique restriction enzyme sites within the 5′-ends of the primers.
- 4. 4 of 7 PCR optimization: PCR reactions are not usually 100% efficient, even when using cloned DNA and primers of defined sequence. It may be necessary to vary the annealing temperature and/or the Mg2+ concentration to obtain faithful amplification. PCR variations: Nested PCR This can be used when the target sequence is known, but the number of DNA copies is very small (e.g. a single DNA molecule from a microbial genome), or if the sample is degraded (e.g. a forensic sample). The process involves two consecutive ‘rounds’ of PCR. The first PCR uses so-called ‘external’ primers, and the second PCR uses two ‘internal’ (or ‘nested(’ primers that anneal to sequences within the product of the first PCR. This increases the likelihood of amplification of the target sequences by selecting for it using different primers during each round. Thus, nested PCR also increases the specificity of the reaction, since a single set of primers used in isolation may give a reasonable yield but several bands, while the use of a second set of primers ensures that a unique sequence is amplified, e.g. in microbial diagnostics. Inverse PCR This is a useful technique for amplifying a DNA sequence flanking a region of known base sequence, e.g. to provide material for characterizing an unknown region of DNA. The DNA is cut with a restriction enzyme so that both the region of known sequence and the flanking regions are included. This restriction fragment is then circularized and cut with a second restriction enzyme with specificity for a region in the known sequence. The now linear DNA will have part of the known sequence at each terminus, and by using primers that anneal to these parts of the known sequence, the unknown region can be amplified by conventional PCR. The product can then be sequenced and characterized. Reverse transcriptase-PCR (RT-PCR) This technique is useful for detecting cell-specific gene expression (as evident by the presence of specific mRNA) when the amount of biological material is limited. Using either an oligo-dT primer to anneal to the 30 polyadenyl ‘tail’ of the mRNA, or random hexamer primers, together with reverse transcriptase, cDNA is produced which is then amplified by PCR. RT-PCR is often a useful method of generating a probe, the identity of which can be confirmed by sequencing. Multiplex PCR: multiple pairs of primers are added, PCR can be used to amplify more than one DNA fragment in the same reaction and these fragments can easily be distinguished on gels if they are of different lengths. This use of multiple sets of primers is often used as a quick test to detect the presence of microorganisms that may be contaminating food or water, or be infecting tissue. PCR with several targets can be monitored by (target-specific) probes labeled with different types of fluorescent dye (i.e. dyes with different emission spectra. As multiple primers are used, extra care is required in order to prevent the formation of primer– dimers. In some cases, one primer can be shared by two targets. For example, a sequence in the 16S rRNA gene in Bacteroides forsythus and Prevotella intermedia has been amplified with one forward primer (a broad-range primer common to both species) and two species-specific reverse primers. Multiplex PCR has been used e.g. for detecting mecA and coa genes in Staphylococcus aureus, for diagnostic virology ؛for detecting toxin genes in Clostridium difficile and for sub speciation of Campylobacter jejuni isolates. Multiplex PCR is also used e.g. for amplification of STRs in human DNA profiling (e.g. CODIS.( An alternative approach is selector-based multiplex PCR.
- 5. 5 of 7 PCR Mutagenesis (Site Directed Mutagenesis) PCR can be used to manipulate DNA. For example, site-directed mutagenesis can be carried out by designing primers with single nucleotide mismatches. Since the primers serve as templates in subsequent rounds of DNA replication the PCR products will contained the introduced nucleotide. Similarly, restriction sites are easily added to the PCR products for subsequent subcloning. Touchdown PCR: In a typical reaction, each of the PCR temperature cycles will differ by 1°C in the annealing temperature and each of these cycles is run twice. The range of temperatures will typically be over 10-20°C (20- 40 cycles) and in the process the touchdown temperature will have been reached and passed. This basic approach may of course be changed in terms of temperature range, the temperature drop and the individual and total numbers of cycles. The rationale of this method is that preference is given to the reaction with the highest Tm (and therefore the highest specificity). Allele-specific PCR: use of an allele-specific primer (and a gene-specific primer) to amplify a particular allele among a mixture of alleles. Asymmetric PCR: a procedure in which the concentration of one primer is much lower than that of the other; it is used for obtaining a particular strand of the template dsDNA. Real-time PCR Conventional PCR techniques rely on end-point detection of amplified product, e.g. by electrophoretic separation and staining. However, such methods are time-consuming and are only semi-quantitative, since they are based predominantly on the detection of an amplified fragment (band) in a sample, rather than being designed to give exact information on its abundance (copy number). Quantitative analysis is only feasible during the early stages of PCR, where reagents are in excess and where the amount of amplified product is small, thereby avoiding the problems of product hybridization, which would compete with primer binding. Real-time PCR A form of PCR in which it is possible to follow the progress of amplification – that is, the ongoing increase in numbers of specific amplicons in the reaction mixture – while it is happening; this approach also permits estimation of the number of specific target sequences that were present in the reaction mixture before the beginning of cycling (one form of quantitative PCR), The increase in numbers of amplicons in a given reaction can be monitored in two main ways: (i) by the use of target specific probes (which give rise to a fluorescent signal in the presence of specific amplicons), and (ii) by the use of certain dyes that exhibit dsDNA-dependent fluorescence, i.e. dyes which fluoresce when they bind to double-stranded DNA (or which increase fluorescence from a minimal to a significant level when they bind to dsDNA). During the reaction, fluorescence is recorded cycle by cycle – beginning at cycle 1 and continuing throughout the reaction to the last cycle (which may be e.g. cycle ~30– 40.( To estimate the number of target sequences present in the mixture prior to cycling it is necessary to determine the so called threshold cycle (Ct, or CT), (i.e. the first cycle in which fluorescence from the reaction exceeds the background fluorescence by a specified amount) The cycle in which significant fluorescence is first detectable is inversely proportional to the amount of initial template. A standard curve can be prepared from this threshold cycle (Ct) and the template concentration of known standards.
- 6. 6 of 7 The threshold cycle allows a quantitative estimation of the initial number of target sequences in the reaction mixture as there is an inverse linear relationship between (i) threshold cycle and (ii) the logarithm of the number of target sequences prior to cycling. That is, a linear graph is obtained – over a range of values – if threshold cycle is plotted against the logarithm of the initial number of target sequences in the reaction mixture. This relationship can be understood intuitively: the lower the number of target sequences present in the reaction mixture – prior to cycling – the higher will be the number of cycles of amplification needed to reach a level of fluorescence corresponding to the threshold cycle (and vice versa). Dye fluorescence This is the simplest and least expensive approach. A fluorescent dye such as SYBR Green is included in the PCR mixture and the level of fluorescence is monitored as the reaction proceeds; since SYBR Green binds strongly to dsDNA, showing an enhancement in fluorescence of over 100-fold, any increase in fluorescence is directly proportional to the amount of dsDNA produced. Calibration is achieved by running a series of standards containing known amounts of dsDNA. This approach works well for optimised single PCR product reactions where non-specific reactions are minimized. Fluorescent reporter probes Here a fluorescent reporter dye is covalently bound to the 50 end of an oligonucleotide probe while a ‘quencher’ group is attached to the 30 end. The probe is designed to hybridize to an internal region of the target sequence. During PCR, when the DNA polymerase molecule reaches the hybridized probe, the 50 nuclease activity of the polymerase will cleave the reporter dye from the rest of the probe, causing an increase in fluorescence with each cycle that is in direct proportion to the amount of PCR product being formed, which is itself directly related to the original number of copies of the target DNA sequence. A commercial example is the TaqMan series of probes – while this approach is more accurate and reliable than dye fluorescence, it is also far more expensive, since a specific reporter probe must be synthesised for each target sequence. Other variants rely on changes in 3D conformation of the probe when it binds to the target sequence, causing an increase in fluorescence, e.g. Molecular Beacons and Scorpion probes.
- 7. 7 of 7 PCR Applications o Diagnosis and screening of genetic diseases and cancer o Rapid detection of slowly growing micro-organisms (e.g. mycobacteria) and viruses (e.g. HIV) o Site-Directed Mutagenesis o Sequencing o Taxonomy o Analysis of DNA in archival material o Gene Expression o DNA fingerprinting in forensic science o preparation of nucleic acid probes o clone screening, mapping and subcloning