In this Guide
Polymerase Chain Reaction
The invention of polymerase chain reaction has proved a revolutionary step for molecular biology. With this extremely powerful technology a short region of DNA can be further amplified with higher order of magnitude to produce thousands to million copies of a specific sequence. The method was introduced by an American biochemist Kary Mullis in the year of 1984. PCR is a technology which has its application in all molecular biology applications. The advent of PCR meant that insufficiencies in the quantity of DNA were no longer a limitation in molecular biology research or diagnostic procedures. The chemistry involved in PCR depends on the complementarity (matching) of the nucleotide bases in the double-stranded DNA helix. When a molecule of DNA is sufficiently heated, the hydrogen bonds holding together the double helix are disrupted and the molecule separates or denatures into single strands. If the DNA solution is allowed to cool, the complementary base pairs can reform to restore the original double helix.
In order to use PCR, the exact sequence of nucleotides that flank the area of interest must be known. This is the absolute minimum data necessary before a typical PCR reaction can be used. This data is necessary for the design of PCR primers that are 5′-3′ oligonucleotides of about 20 nucleotides in length. These are designed to be complementary to the flanking sequences of the target area, as mentioned previously. Thus, the researcher has to either use previous data (known information of sequences) or, if this is unavailable, determine the sequence of these regions experimentally. The two primers (primer pair) can then be synthesized chemically and will then serve as leaders or initiators of the replication step.
The key to the replication reaction is that it is driven by a heat-stable polymerase molecule that reads a template DNA in the 3’-5’ direction and synthesises a new complementary template in the 5’-3’ direction, using free dideoxy nucleoside triphosphates (dNTP’s = nucleotide bases) as building blocks.
Basic principles of PCR methodology involve three steps, namely denaturation, annealing and extension. The method is developed on the basis of DNA polymerization. It uses thermal cycler including repeated heating and cooling mechanisms for melting of DNA and replication using thermo stable DNA polymerase, primer with specific complementary sequence of the target region and dNTPs. All these results in amplification of particular sequence of DNA in the range of upto – 10Kb or in some instances upto -40Kb. In general, the PCR reaction is based on 20-40 repeated cycles with temperature change. At the beginning of the thermal cycle the temperature holds at a high value ( hold temperature, >90 degree Centigrade) which is followed again after the completion of the extension of final products.
The PCR reaction begins with a high temperature of 94–98 °C for an extended period of 1-9 minutes to activate the heat stable DNA polymerase. This step is followed by holding the temperature to .94–98 °C for 20-30 seconds. When a double stranded DNA (dsDNA) molecule is heated to 94oC, the paired strands will separate (denature), by breakage of hydrogen bonding present between complimentary bases, resulting the single stranded DNA formation. This allows the primers access to the single stranded DNA (ssDNA) templates.
In the next step, The reaction mixture is cooled (about 50-65oC) for 20-40 seconds, to allow primers to select and bind (hybridize) to their complementary positions on the ssDNA template molecules. It is important to match the primer sequence with the single stranded DNA closely to form stable hydrogen bond. In principle, the primer sequences are designed to be placed in 3’ region of DNA strand which is supposed to get amplified. As the concentration of primer remains very low in comparison to DNA template, the hybrid formation between primer and template is favoured in respect to re-annealing of DNA template strand.
The elongation step is dependent on DNA polymerase. Taq polymerase shows highest activity at the temperature range of 75-78 oC. The ssDNA/primer solution is thus heated to 72oC. In the presence of the heat stable polymerase, PCR buffer, dNTP’s and magnesium (Mg2+) molecules, the replication procedure begins. At this step, the primers are getting extended towards each other and anneal with the template DNA. With each repetition of this cycle, the target is doubled and soon, after about 30 cycles, the reaction will yield in excess of 1 million copies of the target DNA fragment.
Use of PCR
PCR can be used very effectively to modify DNA. Such modification may include the addition of restriction enzyme sites (in order to facilitate cloning requirements) or regulatory elements (e.g., the addition of promoter sequences to a DNA cistron). A further type of modification can be the generation of desired site directed mutations in a gene, inclusive of sequence alterations, additions or deletions.
Cycle-sequencing, a modification of the classical di-deoxy sequencing method pioneered by Fred Sanger in the early 1980’s, uses the principles of PCR to rapidly perform sequence reactions in a thermal cycler. For PCR-directed diagnostics, it is for example, possible to work with crude samples and minute amounts of material that may include degraded templates, blood, sperm, tissue, individual hairs, etc.
With the advances in molecular biology, the analysis of the genetic material of pathogens has complemented or replaced serological methods for diagnostics, epidemiology and taxonomy. There are significant advantages in the ability to indicate a pathogen’s presence by the detection of its DNA or RNA. Successful bacterial or viral isolation is dependent on the presence of live or viable pathogen in a specimen and is generally time consuming and expensive. It also requires the presence of live pathogen. Antigen detection procedures are limited by the amount and quality of antigen present in specimen. Nucleic acid is more resistant to denaturation than protein and can survive long period of time (even centuries) under appropriate conditions. The limitation on nucleic acid detection has been the very small amounts that are available for detection. Notwithstanding, nucleic acid hybridisation techniques have been used to probe specimens, using a complementary strand of DNA or RNA, appropriately labelled with an enzyme or a radioisotope. Specific base pairing produces a hybrid between the probe and the target, that can be detected based on the label. Such nucleic acid probes have been developed and used for the detection of many pathogens.
PCR represents an entirely new technology. In vitro bacterial or viral culture is widely used to isolate and multiply a pathogen, so that the organism itself, or its antigens, can be more readily detected, by virtue of being present in greater quantity and generally with fewer contaminants. PCR technology permits the same principle (i.e., in vitro amplification) to be applied to the detection of specific sequences of nucleic acid. There are enormous benefits to this approach.
A PCR procedure has been developed to detect Mycoplasma DNA in pleural fluids from cattle with Contagious Bovine Pleuropneumonia (CBPP). PCR was applied and described to the detection of FMD viral nucleic acid in clinical specimens. Primers flanking a conserved region of the polymerase gene were used, so that sequence to any of the virus types could be amplified. Amplicon was detected by agarose gel electrophoresis, on the basis of molecular mass, with confirmation by hybridization to a labelled probe. The procedure was specific for FMD viruses and at least as sensitive as alternative diagnostic procedures. Similarly, a PCR was developed directed towards the detection of FMD virus in oesophageal-pharyngeal tissues and fluids.
A reverse transcriptase method detects and differentiates between rinderpest and Ruminants viruses. Viral RNA is transcribed to cDNA that is then amplified using at least three primer sets. Two sets amplify regions of the fusion protein gene specific for either rinderpest or PPR viruses. The third set is based on a highly conserved region of the phosphoprotein gene that will detect rinderpest and PPR virus and other morbilliviruses. This is included to accommodate the possibility of a small change in the nucleotide sequence of the fusion protein producing a false negative result. The PCR products are resolved on an agarose gel. They are further identified by PCR using nested primer sets based on the amplified fusion protein sequence. Alternatively, the PCR products can be subjected to sequence analysis. PCR can readily be applied to detection of rinderpest and PPR viruses in tears and swabs from eyes, mouth and gum erosions of affected animals. Such samples are readily obtained and easily transported to the laboratory. Furthermore, the PCR product can be used for sequence analysis for epidemiological studies, without the need to first isolate viruses in cell culture.
PCR today plays a central role in genetic typing of organisms or individuals and molecular epidemiology.
To catalyze the amplification, a heatstable DNA polymerase enzyme is used and this enzyme needs to be supplied with an appropriate buffer, metal ion cofactor(s)(for eg. MgCl2– 1.5 mM) and deoxyribosenucleotide building blocks (0.1 µM each) , dNTP’s (100 µM of each of dATP, dCTP, dGTP and dTTP). Other optional components may be added for specific applications (i.e., to stabilise the enzyme, manipulate the denaturing temperatures, etc.). In a “typical” PCR reaction, the following components are mixed in a 0.2 or 0.5 mL PCR tube and made up to 25-100 µL with double distilled (dd) H2O. The reaction mixture is overlaid with 3 drops of mineral oil (i.e., completely cover the surface of the mix with 2 mm oil layer). In the case of PCR machines with heating tops, the mineral oil is not needed.
Temperature profile for the PCR cycles
Any PCR essentially involves a number of cycles at different temperatures. One where the template is denatured (+94°C) for 20 seconds. Longer initial denaturation is usually required (30 s) when working with chromosomal or complex genomic material; another where primers are annealed (hybridised) to the template (wide range of possible temperatures up to 72°C) generally 55°C for a time of 20 – 30 seconds; and a third temperature cycle where primers are extended (72°C for 1 min for every 1000 bp amplicion). In the early days of PCR, tubes were shifted by hand among three different waterbaths or heating blocks that were kept at different temperatures. However, automation has quickly followed and amplification can now conveniently be performed in a DNA Thermal Cycler.
For the many different applications of the PCR, many different methods of isolating and preparing the template DNA exist, mostly depending on the source of the DNA. It is important to note that the outcome of a PCR is dependent on the quality and integrity of the template DNA. It is wise to purify template DNA using a product or method that is specifically designed to purify template DNA for use in PCR. The amount of input template DNA is also of crucial importance in PCR and a common mistake is to add too much template DNA to the PCR reaction. Generally, the amount of DNA per reaction should be 104-106 target/template molecules.
Guidelines for template DNA input
|Human DNA||0.5 mg||1 * 105 targets|
|Blood (Human)||1 mL||7.5 * 104 targets (40 ng)|
|Guthrie blood spot||2.5 mm spot||105 targets|
|Semen||10 ng||3 * 105 targets (5 mg)|
|Yeast||10 ng||3 * 105 targets|
|E. coli DNA||1 ng||1* 105 targets|
|Bacteriophage||1 plaque||1* 106 targets|
Guideline for choosing the number of copies of template
|Number copies starting template||1 Kb DNA||E. coli DNA||Human Genomic DNA|
|10||0.01 fg||0.05 pg||36 pg|
|100||0.11 fg||0.56 pg||360 pg|
|1000||1.10fg||5.60 pg||3.6 ng|
|10000||11.0 fg||56.0 pg||36 ng|
Molar conversion for nucleic acid templates
|1 kb DNA||1000 bp||1.52||9 * 1011|
|Average mRNA||1930||1.67||1 * 10 12|
|E.coli||5 * 10 6*||3 * 10 -4*||2 * 10 8#|
|Human||3 * 10 9||5 * 10 -7||3 * 10 5#|
* Base pairs in haploid genome.
# For single-copy genes.
PCR primers are specific short strings of single-stranded DNA (ssDNA), known as oligodeoxyribonucleotides or oligomers.
These primers flank opposite strands on either end of the target DNA. The design of these primers is very important and it is the composition, sequence match with the template and the concentration of primers that play an important role in the outcome of a PCR assay.
Primer concentrations should be 0.1-0.5 µM in optimal reactions of a standard or basic nature. Higher primer concentrations (> 0.5 µM) may cause the accumulation of nonspecific products by promoting priming at nonspecific sites on the template (mispriming). The primers should nevertheless be in excess in order to avoid their exhaustion before the completion of the reaction that would compromise the yield of the desired amplicon. As a general rule, 5 pmol of each primer / 25 µL PCR reaction, 10 pmol/50 µL PCR reaction and 20 pmol/100 µL PCR reaction.
In order to obtain a molarity of 0.1-0.5 µM, the corresponding molar amount of primer needed in a 100 µL reaction, is 10-50 pmol.
It is useful to dilute oligonucleotide primer stocks to a concentration of 10 pmol/µL (10 µM), enabling the addition of a convenient volume of primer stock to the PCR reaction (1-5 uL/100 µL PCR reaction).
After synthesis, the concentration of the oligonucleotide primer may be expressed in different units, depending on the manufacturer (usually in optical density units (OD), and/or in µg). The following approximate values are useful in calculating your required dilution of the concentrated primer solution.
1 pmol of a 30 mer oligo = 10.26 ng
1 µg of a 30 mer oligo = 0.0975 nmol
1 OD260 (ssDNA) = 33 µg/mL
Design of primers
The design of primers, more than anything else, will determine the success of a specific PCR assay. The two primers are unrelated to one another, since they anneal to different strands and on opposite ends of the target amplicon. However, special care must be taken to ensure that there is no significant complementarity within or between primers and that primer pairs are balanced with respect to the melting temperature (Tm). When two single-stranded DNA molecules hybridize, such as in the annealing of a PCR primer to the template, the stability of the duplex is dependent on the sequence and number of associated base pairs, the concentration of the DNA species, and the salt concentration of the solution. The strands of the duplex can be separated by heat and the temperature at which half the molecules are single-stranded and half are double-stranded, is called the Tm. Thus, the Tm will determine the annealing temperature (Ta) in the cycler temperature profile and should suit both primers. A number of other pointers are also of importance in the design of primers that will function optimally, and will be discussed briefly. All of these criteria can be met in most cases by careful analysis and thought, but for convenience, several primer design software programs are available with most software packages for general DNA sequence analysis.
Applicable annealing temperatures (Ta) are typically 5°C below the true Tm, but optimal annealing temperatures are often higher (5-10°C) than the Tm of the primers.
This optimal annealing temperature is also externally dependent on the salt concentration as well as the Mg2+ concentration in the reaction and has to be determined empirically.
The highest possible annealing temperatures permitted by a specific primer set should be selected since increasing annealing temperatures enhances discrimination and reduces misextension.
Thus a high stringency in the annealing temperature will ensure a good quality product and this should typically be in the range of 55-72°C. Annealing will require only a few seconds at the correct primer concentrations.
The time required for extension of the primers and synthesis of the entire length of the target amplicon, is dependent upon the length and concentration of the target sequence and upon the temperature and the properties of the specific enzyme used. The speed of the cycler and the properties of the tubes used may also be influential, as will later be discussed.
- Extension temperature of 72oC is near the optimal working temperature of most of the heat-stable polymerase enzymes
- Rates of incorporation varies between 35-100 nucleotides/second and therefore, as a general indicator, 1 min at 72°C is sufficient for 2kb products
- Longer cycles may be useful, especially early on if substrate concentration is very low and later on when product concentration exceeds enzyme concentration. A long last cycle (e.g., 10 min at 72°C) is useful to ensure that all amplified copies are full length
Degenerate PCR primers
Occasionally, the exact nucleotide sequence of the target-template DNA will not be known and the sequence has to be deduced from the amino-acid sequence. To enable such templates to be amplified by PCR, degenerate primers can be used. These are actually mixtures of several primers whose sequences differ at the positions that correspond to the uncertainties in the template sequence.
Design and use guidelines
- Avoid degeneracies in the last 3 nucleotides at the 3’ end of the primer
- If possible, use Met or Trp-encoding triplets at the 3’ end
- To increase primer-template binding efficiency, reduce degeneracy by allowing mismatches between the primer and template, especially towards the 5’ end (thus allow primer annealing at lower than optimal Tm). Again, avoid mismatching at the 3’ end
- Design primers with less than 4-fold degeneracy at any given position.
- Increase the primer concentration to 1 µM
Buffer and components
- A buffer that provides ionic strength and buffering capacity during the reaction is required and these aspects of the ionic environment of the PCR are critical
- The standard buffer is usually 10-50 mM Tris-HCl (pH 8.3- 8.8), and up to 50 mM KCl may be included to facilitate primer annealing
- Other buffers used in specific applications may include NaCl (allows better denaturation of GC rich templates) or ammonium sulphate (may limit artifacts due to incomplete amplicons)
- The addition of DMSO can be useful when amplifying multiple sequences in the same reaction (reduce secondary structure of the target DNA), but is generally not recommended because of the inhibitory effect on the enzyme
- Gelatine, bovine serum albumin (BSA), Formamide or Triton X-100 may be included in the enzyme storage buffer by some manufacturers to stabilise the enzyme
The concentration of the essential co-factor, MgCl2, can have a particularly profound effect on the specificity and yield of the PCR.
- The Mg2+ concentration affects the reaction differently at low or high concentrations and is influential in terms of specificity and yield of the amplification reaction
- Mg2+ forms soluble complexes with the dNTP building blocks to make them available and recognisable as substrate for the enzyme
- The concentration of free Mg2+ is determined by the presence and concentration of chelating agents like EDTA, free pyrophosphates, and dNTP’s
The following general factors should be kept in mind:
- It is best to determine the optimal Mg2+ concentration empirically for each PCR assay
- PCR’s should contain 0.5-2.5 mM Mg2+ and t he most commonly used Mg2+ concentration is 1.5 mM, when the four dNTP’s are present at 200 µM each) DNA, dNTPs and proteins will bind to Mg2+
- Total [Mg2+] = (bound + free) [Mg2+]
- Free [Mg2+] should equal the [dNTP]
- Presence of EDTA, hemoglobin, heparin and other chelators in the PCR reaction mixture may influence the apparent [Mg2+]
- It is preferred that blood samples be collected in the presence of citrate. The second best is EDTA blood-tubes, but heparin tubes should never be used
- Self-adjusting magnesium buffers that automatically adjust the Mg2+ concentration throughout the PCR reaction can also be used.
A working stock containing l0 mM of each dNTP (pH7.0) is recommended and a final reaction concentration between 20 and 200 µM each (mostly depending on amplicon size, [Mg 2+ ]), result in the optimal balance in yield, specificity and accuracy. This molarity represent a sufficient excess to allow for the concentration of dNTP’s to remain virtually constant throughout the reaction. Higher dNTP concentrations lead to a decrease in the specificity and fidelity of PCR. It is important that dNTP’s should be used at equivalent concentrations (i.e., should be balanced) in order to minimize misincorporation errors. In general: 200 µM of each dNTP/2 kbp amplicon @ 30 amplification cycles
Enzymes and enzyme concentration
The recommended polymerase concentration is usually 1-2.5 Units per 100 µL reaction volume (most often supplied as 5U/µL). This would represent the comparatively low molar equivalent of 1-2.5 nM, relative to all the other reaction components. This limiting concentration ensures fidelity since higher enzyme concentrations result in nonspecific background products. When using other additives in reactions, their effects on the enzyme should be kept in mind. Glycerol for example increases thermal stability of the enzyme but reduces the Tm. DMSO also lowers Tm but reduces thermal stability of the enzyme (reduced Tm allows lower temperature and therefore less DNA damage). The polymerase is a complex molecule with three activities; a 5’3’ polymerase activity, a 3’-5’ exonuclease activity (proofreading activity) and a 5’-3’ exonuclease activity.
Optimizing a PCR application
Some commercial companies also offer a PCR optimisation kit that may be used to simplify the PCR optimisation procedure. In addition, approaches such as the touchdown PCR also offers simple onestep o ptimization of PCR reactions that are expected to be sub-optimal with regard to primer/template homology. As a general rule however, any PCR that will become an established assay in the laboratory should be properly optimised by a titration method. We like to recommend a most useful simplified protocol for PCR optimisation, based on a set of methods that are widely applied development trials for industrial process design, the so-called Taguchi methods.
Detection of Newcastle disease virus using a Triple One-step RT-PCR Assay
Viral RNA is extracted from sample material, then converted into cDNA using reverse transcriptase and amplified thereafter by a Taq DNA polymerase (one-step RT-PCR).
Three tests can be done using three primer pairs, that will detect:
- Presence of virus
- Presence of virulent virus
- Presence of avirulent virus
The amplified DNA is then visualised by size fractionation on an agarose gel and the results recorded by using a photo documentation system.
All laboratory personnel must be familiar with the extraction and handling of RNA, and all procedures needed to perform RT-PCR.
Micropipettes: 10 µL; 100 µL; 1000 µL
Micropipette filter tips: 10 µL, 100 µL, 1000 µL
Microcentrifuge tubes (2.0 mL)
Heating block 55oC
Biohazard flow cabinet (N/A 6 ft LF – Class II)
Laminar flow cabinets
Material and Reagents
dNTPs 10 mM (Lab. Spec.)
Oligonucleotides – ALLs/ALLe/VLTe/AVLe 20 pmol/ µL
DNA polymerase: 250 U HotStar Taq DNA polymerase
500 U FastStart Taq DNA polymerase
MMLV-RT 200 U / µL (e.g., Promega)
RNasin Ribonuclease Inhibitor 40 U/µl (e.g., Promega)
Chloroform (e.g., Merck)
Absolute ethanol (e.g., Merck)
Isopropanol (e.g., Ass. Chem. Enterprises)
RNase-free H2O (e.g., USB)
1 x TAE buffer
Agarose (e.g., Separations)
DNA molecular weight marker
QiaAmp Viral RNA Isolation Kit (Qiagen) 250 reactions
TRI Reagent (Sigma)
Control Reference Material Test controls
- Allantoic fluid from embryonated SPF chicken eggs infected with NDV (see preparation protocol)
- A high titre positive sample
- A low titre positive sample (or the above sample suitably diluted)
- NDV cDNA
- Tracheal swabs from chickens/ostriches infected with NDV (if available)
- Allantoic fluid from NDV-negative embryonated SPF chicken eggs.
- Allantoic fluid from embryonated SPF chicken eggs infected with another virus, e.g., avian influenza (if available)
Performance of assay
Ostrich tracheal swabs
- Collect a copy of the registration form on the sample reception board
- Collect samples from -70°C freezer
- Leave at room temperature (18-25oC) in Lab 2 to thaw
- Pool 5 samples derived from the same farm into one microcentrifuge tube, using 50 µL from each
- Return original samples to -70°C freezer
- Proceed with either the TRI Reagent or QiaAmp extraction method
For other NDV samples (allantoic fluid)
- Collect a copy of the registration form on the sample reception board
- Collect samples from the -70°C freezer
- Leave at room temperature (18-25oC) in Lab 2 to thaw
- Use 250 µL from each sample separately
- Return original samples to -70°C freezer
- Proceed with either the TRI Reagent or QIAamp extraction method
- Add 1mL of TRI Reagent to 250 µL allantoic fluid in a microcentrifuge tube
- L eave on ice for 5 min
- Add 200 µL of chloroform and vortex.
- Centrifuge at 18000 g for 15 min at 4oC
- Transfer the upper aqueous phase to a clean microcentrifuge tube and add 600 µL of isopropanol
- Precipitate at –20°C for a minimum time of 20 min or overnight
- Centrifuge at 18000 g for 15 min at 4oC and discard the supernatant
- Add 900 µL of 70% ethanol and invert gently to wash the pellet
- Centrifuge at 18000 g for 10 min at 4oC
- Discard the supernatant and air-dry the pellet in a laminar flow cabinet for 10 min. Do not let the RNA pellet dry completely
- Dissolve the pellet in 30 µL DEPC-H2O / RNase-free H2O 12. Incubate at 55°C for 10 min and store at -20°C
QIAamp extraction kit
- Add 560 µL prepared AVL/Carrier RNA to 140 µL allantoic fluid in a microcentrifuge tube
- Mix by pulse-vortexing for 15 s and incubate at room temperature (18-25oC) for 10 min
- Add 560 µL absolute ethanol and vortex
- Briefly centrifuge to remove drops from the inside of the lid
- Carefully apply 630 µL of the solution to the spin column (in a 2 collection tube) and centrifuge at 6000 g / 8000 rpm for 1 min
- Place the spin column into a clean 2 mL collection tube and discard the previous collection tube containing the filtrate
- Repeat the previous two steps with the remaining solution
- A dd 500 µL AW1 buffer to the spin column and centrifuge at 6000 g/8000 rpm for 1 min
- Place the spin column into a clean 2 mL collection tube and discard the previous collection tube containing the filtrate
- Add 500 µL AW2 buffer to the spin column and centrifuge at 20 000 g /14 000 rpm (at full speed) for 3 min
- Place the spin column into a clean microcentrifuge tube and discard the previous collection tube containing the filtrate
- Add 60 µL elution buffer and incubate at room temperature (18-25oC) for 1 min.
- Centrifuge at 6000 g/8000 rpm for 1 min
- Discard the spin column and store the RNA at -20°C
Preparation of solutions QIAamp Reagents
- Add 1 mL AVL buffer to Carrier RNA
- Dissolve Carrier RNA thoroughly and transfer to the AVL buffer bottle
- Make 1 mL aliquots and store at 4°C
- Incubate at 80°C in a heating block for not more than 5 min before use
Points to consider for PCR reaction
For a successful completion of PCR reaction, several factors play critical role. Such as-
DNA template- As a template DNA obtained from sources like genomic DNA or gDNA, complementary DNA or cDNA, and plasmid DNA is used. DNA composition and complexity is thus important for successful amplification. During a 50 µL PCR reaction, plasmid DNA amount of 0.1–1 ng is enough for amplification process, while in comparison a 5–50 ng of DNA is required if the DNA source is genomic. When necessary, PCR product is also used as template and re-amplifies to increase the yield of final product. Further use of diluted reaction components like primers, dNTPs or salts are highly recommended to avoid the inhibitory effect of un purified PCR reaction materials.
DNA polymerase- DNA polymerase is critical for amplification of target DNA sequence. Among the other DNA polymerase Taq DNA polymerase is most widely used for PCR amplification process. Taq DNA polymerase shows high thermo stability and longer half-life of (approximately 40 mins at a temperature of 95°C.) It has the ability to incorporate almost 60 bases per second at a temperature of 70 °C and is able to amplify the DNA up to 5 Kb lengths. For a standard 50 µL PCR reaction, 1-2 units of Taq DNA polymerase is used to amplify the target sequence.
Primers- synthetic DNA oligonucleotides with s length of 15-30 bases are used as PCR primers. During PCR amplification process, primers are are able to complimentary bind to the flanking region of DNA of interest. While designing PCR primers, in addition to sequence homology, several other factors must be in consideration. Such as-
- A melting temperature in the range of 55–70°C is desirable.
- To avoid primer dimer formation, primers are required to designed in such way that complimentary bond formation can be avoided.
- Additionally, GC content of primers is required to be 40-60% with a uniform distribution of GC bases. Next to minimize non-specific primers, presence of not more than three G, C bases at the 3’ primer end.
- Longer primers with greater than 60 nucleotides are required to purify unconjugated nucleotides or products which are not full length.
- Primers are normally used in the concentration of 0.1–1 μM, whereas a higher concentration of 3–1 μM is required for primers with bases with degenerate in nature.
Deoxynucleoside triphosphates (dNTPs)- In a PCR reaction, equimolar amount of basic nucleotides namely dATP, dCTP, dGTP, and dTTP are added. For a normal PCR reaction individual DNTPs in a final concentration of 0.2 mM. is recommended. In some instances, to prevent the PCR contamination carryover, DNTPs get substituted by special nucleotides. When deoxyuridine triphosphate (dUTP) is substituted for dTTP produces PCR products with uracil. Thus a pre-incubation step with uracil DNA glycosylase or UDG will be helpful to remove uracil containing PCR reaction carryovers resulting in lower false positive results.
Magnesium ion (Mg2+)- Magnesium ion helps in DNTPs incorporation at the time of polymerization reaction. During PCR reaction, mostly MgCl2 solution is the source for Mg2+ ions. However, polymerases like Pfu DNA polymerase used MgSO4 as a source for Mg2+ions. In normal PCR reactions, Mg2+ ion in a concentration of 1–4 mM is used. Use of low amount of Mg2+ ions lowered the activity polymerase activity which in turn decreases PCR product formation. Additionally higher Mg2+ ion concentration produces nonspecific product due to higher primer.-template stability.
PCR Buffer- A buffer with a pH range of 8.0 to 9.5 is usually recommended to stabilize the DNA polymerase activity. A common buffer for using Taq DNA polymerase is K+ ion is KCl resulting in annealing of primer. In some instances, ammonium sulfate or (NH4)2SO4 can be used in place of K+ ion.
What are the different PCR mechanisms?
In RT-PCR method, RNA molecules are used as template. RNA is get converted to complementary DNA or cDNA with the help of reverse transcriptase. For a successful completion of RT-PCR ,maintenance of RNA quality and quantity is important. The method starts with hybridization of DNA/ RNA. Reverse transcriptase enzyme also known to possess and RNase H activity which is able to degrade the RNA from DNA/RNA hybrid. The single stranded molecule of DNA is then used to convert to cDNA by DNA dependent polymerase function of RNaseH The rate at which first strand is synthesized is important for the RT-PCR method. Next standard amplification method is used for cDNA amplification. cDNA being more stable than RNA. RT-PCR method working as the first step for qPCR, quantifying RNA transcripts level in biologically originating samples.
qPCR and RT-qPCR
qPCR or Quantitative PCR is useful for the detection, characterization and quantification of nucleic acid In qPCR, DNA get converted to cDNA followed by quantitative amplification by PCR. In comparison to standard DNA PCR steps like denaturation, annealing and elongation, a florescent labelling method gives the chance to follow the final products. qPCR method which is based on dye ( florescent) , the dye get bound with double stranded DNA and allow to quantify the amount of DNA in every cycle through measuring the fluorescence signal. The term’ real time’ denotes the amount of fluorescence signal quantified is proportional to the amplified DNA. On the other hand, in probe dependent qPCR methods, several targets are identified from a sample at a same time but requires prior target specific probe designing and optimization. Among the different kind of probes used, hydrolysis prob which incorporates fluorophore and quencher are most widely used. When the probe is intact, fluorophore emission via quencher is inhibited by Fluorescence resonance energy transfer (FRET). While at the time of PCR amplification, at the time of primer extension and amplification, the probe get hydrolysed. As a result, fluorophore get separated from quencher which can be determined as increased fluorescence proportional to amplified product.
Hot start PCR
Hot start PCR is an alternative method for reducing non specificity of amplification by setting the amplification process at room temperature. Hot start PCR is suitable for low abundance target amplification including genes which are present in single copy. In general DNA polymerase is quite thermostable and remain active in room temeperature also resulting in random primer association. In contrary, Hot start enzymes, remain inactive at room temperature and only heating is able to activate them. As a result primer-dimer formation as well as extension of misannealed primer extension get reduced resulting in lower PCR background.
Long range PCR
Application such as physical mapping or direct cloning of genome requires amplification of large DNA fragments. Conventional PCR can perform well for amplification short segments of DNA (usually upto 5Kb), while amplification efficiency decreases for long DNA fragments as enzymes such as Taq DNA polymerase lacks the 3’-5’ exonuclease activity required for DNA proofreading.. To circumvent this problem and to amplify larger DNA fragments a mixture of two thermostable DNA polymerase is used. While the first polymerase lacks the proofreading activity, the second DNA polymerase present in lower concentration supported with 3’-5’ exonuclease activity required for potent proofreading. The misincorporation of dNTPs by non-proofreading enzyme thus get removed by proofreading enzymes such as Pfu DNA polymerase allowing amplification of long DNA sequence.
In-situ PCR is a novel method combining sensitivity of polymerase chain reaction with histological location provided by the process of in-situ hybridization. This method is used for the identification and localization of rare or single copy number sequence of DNA or mRNA in frozen tissue sections. This method is based on histology and directly detects DNA or mRNA in tissue samples. Nucleic acid probes such as radiolabelled or fluorescent label is used for hybridization. In situ PCR method involves fixation of tissue and subsequently digestion to give the PCR reagents access towards the DNA or mRNA present in tissue. Target DNA sequence further get amplified by PCR reagents and detected with standard imunocytochemical methods. This method is highly sensitive, amplifying in-situ 150–350 bp DNA for the gene of interest. In-situ PCR has several advantages such as microscopic identification of cells carrying a specific gene in silent form in tissue section.
To promote specificity of conventional PCR, touchdown PCR is used. During the initial cycles of PCR reaction, the annealing temperature set higher than the primer melting temperature ( Tm), resulting in reduced primer-dimer and nonspecific interaction between primers and DNA template. However, increased annealing temperature may lead to lower amount of PCR products. To reduce this effect, the annealing temperature mostly reduced initially at each cycle almost 1°C for the sufficient production of amplicon. In following cycles, once the annealing temperature increased to optimal temperature, usually 3-5°C lower than the Tm of primer it is supposed to be’ touches down’. As a result, final PCR products get increased with less nonspecificity.
Nested PCR is used to increase the productivity of the conventional PCR. This methods involves two set of primers namely outer primer and nested primer. The outer primers covers the flanking region present outside of the DNA to be amplified. While the nested primer covers the exact region of DNA under amplification. At the first round of PCR, the target DNA is amplified together with the flanking region and second round PCR using nested primer used this product as template. If a nonspecific region get amplified by first round primer, it is less likely to further amplification of the nonspecific region by second primer set. This nested primer helps to reduce nonspecific target accumulation as well as increased production of target.
In Fast PCR, the number of PCR steps gets reduced without affecting the yield of PCR product through the use of DNA polymerase with high processivity. Highly proecssive DNA polymerase is able to incorporate a larger number of nucleotides during each cycle providing higher efficiency in lower time compared to conventional PCR. Generally Fast PCR takes 1/2 to 1/3 lesser time than conventional PCR with Taq polymerase. Further amplification time get shortened with the combination of primer annealing an d extension in a single event known as two-step PCR. Low processive DNA polymerase such as Taq polymerase can be used in fast PCR for shorter amplicon size such as less than 500bp.
Direct PCR performs DNA amplification of target region directly instead of isolated nucleic acids. During this method, samples like tissue or cells are used to get lysed in special buffer to release the DNA in presence of high denaturation steps. Direct PCR provides advantages over conventional PCR due to shorter time frame and lowered DNA loss which results from purification stages. In general to avoid inhibition of direct PCR caused by cellular debris, proteins, lipids or polysaccharides use of high processivity enzymes are recommended. As this enzymes are able to amplify smaller DNA amount from unpurified origin with higher sensitivity.
DNA templates with high GC content provide difficulties in amplifying due to the presence of strong hydrogen bond between G and C nucleotide. Additionally higher GC containing region also generates secondary structures. Thus to reduce strong interaction of GC region, PCR additives are often used. PCR additive such as DMSO is able to DNA denaturation and lowering primer Tm. Highly processive enzymes are recommended for GC rich PCR, as they are to bind strongly to the template at the time of primer extension.
Multiplex PCR methods is a single tube simultaneous detection methods of several targets This method gives advantages over conventional PCR as it require less time, small amount of reagent as well as comparison of several different amplicon is possible. However, nonspecificty due to presence of different length PCR primers is an issue of multipley PCR. Therefore prior optimization of primer pairs are essential for multiplex PCR. Primer pairs with the Tm variation within 5 °C for each are generally recommended. Before going to multiplexing events individual primers are required to validate through single plexing amplification for the detection of primer sensitivity and specificity. Additionally different length amplicons are required to get resolved by gel electrophoresis. Further use of Hot start enzymes and specially formulated buffer for multiplexing can increase the product formation.
Inverse PCR is useful for the detection of unknown adjacent regions such as promoter region of a gene, chromosomal rearrangements like gene fusion, translocation and integration of viral gene in host genome. The primers are designed to get further apart from each other rather coming towards and thus named as inverse PCR. In general inverse PCR is widely used in site directed mutagenesis approach. Before beginning inverse PCR, the PCR amplicon get digested by restriction enzymes followed by ligation. Next inverse PCR takes place with primers designed on the basis of known genomic region. As the PCR products contains partly known regions, further sequencing from the two ends of the amplicon result in the identification of adjacent unknown regions. Restriction enzymes are chosen not to digest the known region and ligation is required to take place between adjacent region which is flanking in both sides of known DNA region.
What are theromstable DNA polyemerases ?
Invention of thermostable DNA polymerases is known as a revolutionary steps behind the popularity of PCR as they are able to withstand high denaturing temperature. As a result, use of high annealing temperature can be set with higher stringency for primer annealing. Thenrmostable DNA polymerases further divided on the basis of exonuclease activity namely on proof reading vs non proof reading DNA polymerases.
Taq DNA Polymerase- Taq DNA Polymerase is most widely used DNA polymerase which has been isolated from bacteria Thermus aquaticus. Taq DNA Polymerase functions in catalyzing the incorporation of nucleotides depending on primer and Mg2+ presence. This enzyme mostly works well for DNA and RNA sequence detecting which do not need the presence of high fidelity enzymes. The error rate for this enzyme is almost 1 × 10–5 errors/base. Taq DNA Polymerase works well for the amplification of DNA sequence upto of 5kb. However, for longer PCR products more optimization steps are necessary. Taq DNA polymerase falls under the group of processive enzyme which can incorporate almost 60 nucleotides per second at a temperature of 70°C and at a temperature of 95°C, the half life is 40 minutes. Due to absence of proof reading activity, Taq DNA polymerase produces a 3’ overhang with single base.
Tfl DNA Polymerase- Tfl DNA Polymerase function in similar way like Taq DNA Polymerase without 3′→5′ exonuclease activity. In presence of Mg2+, this polymerase catalyzes the nucleotide polymerization in duplex DNA. In contrary, Tfl DNA Polymerase requires Mn2+ ion in case of RNA template
Tli DNA Polymerase- Tli DNA Polymerase function in DNA polymerization in the direction of 5′→3′ . It also shows 3′→5′ exonuclease activity resulting in increased in fidelity at the time of nucleotide incorporation. On the other hand this exonuclease activity also results in nucleotide degradation required for DNA synthesis initiation. However this could be prevented by methods like hot start PCR. Mostly Tli DNA Polymerase used in PCR, RT-PCR or long PCR.
Pfu DNA Polymerase Pfu DNA Polymerase is known to show lowst error rate among the thermostable enzymes due to presence of highly effective 3′→5′ exonuclease activity. This enzyme mostly used in cloning and DNA expression following PCR. In addition Pfu DNA Polymerase is used alone or in combination with enzymes lacking proofreading activity.
Modification of PCR for specific applications
In several instances, PCR amplification required to be modified. Such as-
Cloning- During cloning procedure, a sequence of interest is get amplified through PCR and then inserted into a vector. These particular DNA fragments can be further modified to introduce specific enzyme recognition regions. Further primer pair can be designed to amplify cloned fragments from the vector with the help of high fidelity enzyme for low error.
Sequence Tag Sites- Sequence Tag Sites are required at the time of high through output sequencing technologies. Tagged sites are mostly functions in to identify the presence of specific sequence in a clone and thus the data for genomic sequence can be related to the original source.
Site-directed Mutagenesis- During the study of functional aspects of proteins, sometimes a mutation is generated in the genomic sequence. This is mostly done by incorporating base change in the primer, generally in 5’ site also including restriction enzyme sites for further cloning.