Polymerase Chain Reaction

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.

PCR principles

Denaturation

When a double stranded DNA (dsDNA) molecule is heated to 94oC, the paired strands will separate (denature). This allows the primers access to the single stranded DNA (ssDNA) templates.

Annealing 

The reaction mixture is cooled (about 50oC) to allow primers to select and bind (hybridize) to their complementary positions on the ssDNA template molecules.

Elongation

The ssDNA/primer solution is heated to 72oC. In the presence of the heat stable polymerase, PCR buffer, dNTP’s and magnesium (Mg2+) molecules, the replication procedure begins. 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.

Basic reactions

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.

Template DNA

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

Nucleic acid size pmol/mL Molecules/mg
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.

Primers

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 concentration

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.

Calculations

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.

Formulae

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.

Primer annealing

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.

Primer extension

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

Magnesium concentration

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.

dNTP’s

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

Background principle

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:

  1. Presence of virus
  2. Presence of virulent virus
  3. 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.

Pre-conditions

All laboratory personnel must be familiar with the extraction and handling of RNA, and all procedures needed to perform RT-PCR.

Procedure

Equipment  

Micropipettes: 10 µL; 100 µL; 1000 µL Micropipette filter tips: 10 µL,  100 µL,   1000 µL Microcentrifuge tubes (2.0 mL) Heating block 55oC Microcentrifuge Vortex apparatus Biohazard flow cabinet (N/A 6 ft LF – Class II) Laminar flow cabinets Thermocycler Microwave oven Gel apparatus Gel analyser Ice machine

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) 70% Ethanol DEPC-H2O RNase-free H2O (e.g., USB) 1 x TAE buffer Agarose (e.g., Separations) DNA molecular weight marker Loading buffer QiaAmp Viral RNA Isolation Kit (Qiagen) 250 reactions TRI Reagent (Sigma) Control Reference Material Test controls

 Positive 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)

Negative controls

  • Allantoic fluid from NDV-negative embryonated SPF chicken eggs.
  • DEPC-H2O
  • Allantoic fluid from embryonated SPF chicken eggs infected with another virus, e.g., avian influenza (if available)

Performance of assay

Sample preparation

Ostrich tracheal swabs

  1. Collect a copy of the registration form on the sample reception board
  2. Collect samples from -70°C freezer
  3. Leave at room temperature (18-25oC) in Lab 2 to thaw
  4. Pool 5 samples derived from the same farm into one microcentrifuge tube, using 50 µL from each
  5. Return original samples to -70°C freezer
  6. Proceed with either the TRI Reagent or QiaAmp extraction method

For other NDV samples (allantoic fluid)

  1. Collect a copy of the registration form on the sample reception board
  2. Collect samples from the -70°C freezer
  3. Leave at room temperature (18-25oC) in Lab 2 to thaw
  4. Use 250 µL from each sample separately
  5. Return original samples to -70°C freezer
  6. Proceed with either the TRI Reagent or QIAamp extraction method

RNA Isolation

TRI Reagent

  1. Add 1mL of TRI Reagent to 250 µL allantoic fluid in a microcentrifuge tube
  2. L eave on ice for 5 min
  3. Add 200 µL of chloroform and vortex.
  4. Centrifuge at 18000 g for 15 min at 4oC
  5. Transfer the upper aqueous phase to a clean microcentrifuge tube and add 600 µL of isopropanol
  6. Precipitate at –20°C for a minimum time of 20 min or overnight
  7. Centrifuge at 18000 g for 15 min at 4oC and discard the supernatant
  8. Add 900 µL of 70% ethanol and invert gently to wash the pellet
  9. Centrifuge at 18000 g for 10 min at 4oC
  10. Discard the supernatant and air-dry the pellet in a laminar flow cabinet for 10 min. Do not let the RNA pellet dry completely
  11. 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

  1. Add 560 µL prepared AVL/Carrier RNA to 140 µL allantoic fluid in a microcentrifuge tube
  2. Mix by pulse-vortexing for 15 s and incubate at room temperature (18-25oC) for 10 min
  3. Add 560 µL absolute ethanol and vortex
  4. Briefly centrifuge to remove drops from the inside of the lid
  5. 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
  6. Place the spin column into a clean 2 mL collection tube and discard the previous collection tube containing the filtrate
  7. Repeat the previous two steps with the remaining solution
  8. A dd 500 µL AW1 buffer to the spin column and centrifuge at 6000 g/8000 rpm for 1 min
  9. Place the spin column into a clean 2 mL collection tube and discard the previous collection tube containing the filtrate
  10. Add 500 µL AW2 buffer to the spin column and centrifuge at 20 000 g /14 000 rpm (at full speed) for 3 min
  11. Place the spin column into a clean microcentrifuge tube and discard the previous collection tube containing the filtrate
  12. Add 60 µL elution buffer and incubate at room temperature (18-25oC) for 1 min.
  13. Centrifuge at 6000 g/8000 rpm for 1 min
  14. Discard the spin column and store the RNA at -20°C

Preparation of solutions QIAamp Reagents

Carrier/RNA

  1. Add 1 mL AVL buffer to Carrier RNA
  2. Dissolve Carrier RNA thoroughly and transfer to the AVL buffer bottle
  3. Make 1 mL aliquots and store at 4°C
  4. Incubate at 80°C in a heating block for not more than 5 min before use