Molecular Biology: PCR techniques
Molecular Biology:
PCR Techniques
Author: Prof Estelle Venter
Licensed under a Creative Commons Attribution license.
Table of Contents
INTRODUCTION 2
The principle of PCR 2
MATERIALS AND METHODS 5
Components needed 5
The different steps in PCR 6
CONTAMINATION 12
OTHER PCR’s 13
Reverse transcription PCR 13
Random amplification of polymorphic DNA 14
Multiplex PCR 14
Nested PCR 14
Touchdown PCR 14
Hot Start PCR 15
Real-time PCR 15
(DIAGNOSTIC) APPLICATION OF PCR 15
THECHNIQUES USED IN PCR DIAGNOSTICS 17
FAQs 18
REFERENCES 20
INTRODUCTION
The polymerase chain reaction (PCR) is a simple method for producing unlimited copies of a specific DNA sequence in a test tube which allows a “target” DNA sequence to be selectively amplified several million-fold in just a few hours. The PCR achieves amplification of a predetermined fragment of DNA, (the target; which can e.g. be from 100 – 1000 bp long) with the apparent disadvantage that the sequences flanking the target region must be known, the latter precludes the use of PCR from analysis of DNA regions that have not previously been studied by standard methods.
The principle of PCR
The 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. High temperatures are used to separate the DNA molecules into single strands, and the synthetic sequences of ss DNA (18-30 nucleotides) serve as primers. One primer is complementary to the one DNA strand at the beginning of the target region; a second primer is complementary to a sequence on the opposite DNA strand at the end of the target region.
5’-TTAACGGGGCCCTTTAAA..target sequence..TTTAAACCCGGGTTT-3’ Positive DNA strand
5’-TTAACGGGGCCCTTTAAA-3’...... >Primer 1
and:
<...... 3’-AAATTTGGGCCCAAA-5’ Primer 2
3’-AATTGCCCCGGGAAATTT..target sequence..AAATTTGGGCCCAAA-5’ Negative DNA strand
Location of PCR primers
These are extended towards each other by a thermostable DNA polymerase in a reaction cycle of three steps: denaturation, primer annealing and extension/polymerization/extension.
Figure 2. Location of primers in a PCR (Brown, 1995)
Figure 3. The three steps of the PCR
MATERIALS AND METHODS
Components needed
Template
Any source that contains one or more intact target DNA molecules can be amplified by PCR. Many different methods of isolating and preparing the template DNA exist; however, the extraction method chosen does depend on the source of the DNA. Sources of DNA include e.g. blood, sperm or any other tissue, old forensic specimens, ancient biological samples or in the laboratory, bacterial colonies or phage plaques as well as purified DNA. PCR can only be applied if some sequence information is known so that primers can be designed (PCR Applications Manual, Boehringer Mannheim1995).
Primers
Primers are pairs of oligonucleotides of about 18-30 nucleotides and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures.
Deoxynucleotide triphosphate (dNTP)
A generic term referring to the four deoxyribonucleotides:
dATP, dCTP, dGTP, dTTP
Enzyme
Polymerases are normally used to amplify DNA. Taq polymerase is a unique thermostable enzyme used in the PCR. This enzyme will not denature at 95 °C and will work optimally at 72 °C.
Reaction buffer
The reaction buffer is a buffer especially prepared for the enzyme to work optimally. Most of the reaction buffers are supplied as a 10 x stock solution. The buffer should be diluted to 1 x in the reaction cocktail, 1:10 (v/v). Use the recommended buffer that is supplied with the specific enzyme. Read the product information sheets that are supplied with the enzymes.
Thermocycler
A machine that can change the incubation temperature of the reaction tube automatically, cycling between approximately 95 – 98 °C (for denaturation), 55 - 65 °C (for oligonucleotide annealing, depending on the sequence of the primers) and 72 °C (for synthesis)
The different steps in PCR
Obtaining the template - Isolation of DNA or RNA
The first step in any PCR is to isolate the nucleic acid to be amplified, the template, from the sample. DNA is required principally for two reasons: to enable gene banks to be made and for analysis of the genome, most often with respect to an individual gene that is sought or has already been isolated. RNA and in particular messenger RNA (mRNA) can also be isolated, cDNA can be synthesized and cloned to make a cDNA library.
The primary aim of any nucleic acid isolation procedure is to inactivate endogenous nucleases as soon as possible after the intact cell is lysed, and then to free the nucleic acid completely from adhering protein and other macromolecules.
The isolation of DNA and RNA can be illustrated as follows:
· DNA is chemically stable and not susceptible to enzymatic degradation. Isolations are performed at room temperature. In contrast, RNA is chemically unstable and is easily degraded by omnipresent and persistent RNases. RNA is therefore isolated as many enzymes: as fast as possible and at low temperature. The procedures depend on the source, but most protocols contain the following steps (Roche Molecular Biochemical’s. PCR Applications Manual, 1999; PROMEGA: Protocols and applications guide 1996).
· Cells or tissues are lysed; (1) enzymatically by the proteolytic enzyme proteinase K in the presence of sodium dodecyl sulfate (SDS), or (2) chemically by guanidinium isothiocyanate (GITC). Lysis of the cell, dissociation of much of the protein and rapid denaturation of degradative enzymes can be accomplished by a single chemical, the anionic detergent sodium dodecyl (also called lauryl) sulphate (SDS), or its close relative sodium lauroyl sarcosinate. Sometimes e.g. for bacterial and plant cells but not for protozoa and animal cells, degradative enzymes such as lysozyme should be added. Nematodes and adult worms need even more harsh conditions: freezing and thawing, hypochlorite and sonication.
· Removal of proteins by extraction with phenol and or chloroform
· Precipitation of DNA by ethanol and washing the precipitate to remove detergents, salts etc.
· Dissolving the DNA in TE, neutral 10 mM Tris/HCl buffer with 1 mM EDTA to bind Mg2+ and Ca2+ ions that act as cofactors of most nucleases.
Figure 4. Phenol extraction and alcohol precipitation of DNA
Phenol extraction and ethanol precipitation can often be replaced by binding DNA to glass particles or special resins. After washing the particles with an ethanol-containing buffer, the DNA can be eluted by TE.
For the isolation of RNA, contaminating DNA can be removed by centrifugation, acid phenol extraction or RNase-free DNase. Many special procedures exist for the isolation of plasmid DNA from transformed bacteria (like quick-and-dirty minipreps) and for the isolation of DNA from agarose gel.
If DNA is to be used for PCR no extensive purification is required. Moreover, only a small amount of DNA is sufficient to start the amplification. However, contamination with DNA from other sources may cause misleading results. Typical quantities: 1 ml of human blood yields 20 to 50 µg DNA.
DNA embedded in agarose
DNA can be extracted from an agarose gel. This can be either restriction fragment segments or PCR products. The segment is usually cut from the gel with a scalpel blade. One should be very careful in doing this. Many DNA purification methods/kits exist to clean the DNA from the gel.
The amount of template DNA added to a PCR should be:
One typically measures DNA quantity in ng, but the relevant unit is actually moles, i.e., how many copies of the sequence that will anneal with your primers are present. Thus, the amount of DNA in ng that you need to add is a function of its complexity (http://irc.igd.cornell.edu/Protocols/PCR_principles.htm)
· 25-50 ng eukaryotic genomic DNA in a 50 µl total volume reaction
· 0.5 ng plasmid DNA
· 1 µl of boiled bacterial overnight culture (too much inhibits the reaction)
· For re-amplification of a PCR product: 1 µl or less of the primary PCR product
If you suspect that the sample contains inhibitors of the reaction:
· Dilute the sample 1:10 or 1:100
· Test the inhibition by adding an aliquot of the samples to the positive-control sample
Primers
Primers are pairs of oligonucleotides of about 18-30 nucleotides and have similar G+C contents so that they anneal to their complementary sequences at similar temperatures (Dieffenbach and Dveksler 1995).
When designing PCR primers, the following should be taken in consideration:
· Primers should be 18 – 30 nucleotides long
· The target sequence should be 100 – 1000 bp (with 5000 bp as a practical limit)
· There should be a balanced distribution of G/C and A/T rich domains
· Primers should have 10 – 12 Gs or Cs and a Tm (melting temperature) of at least 60 °C; a rough estimate Tm = 4 x (number of G + C) + 2 x (number of A + T). The calculated Tm (melting temperature) for a primer pair should be balanced. Rule of thumb: Tm = 4(G+C) + 2(A+T) and –1.5 °C for every mismatch. A Tm of 55-80 °C is desired
· Primers should not form secondary structures
· Primers should not end with AAA –3’, and GGG-3’, etc. (with eukaryotes also avoid the microsatellite motifs CACA and TGTG)
· Primers should not form dimers. Dimers are formed by primer molecules that can hybridize to each other because of complementary bases in their sequences. Such primer dimers may be elongated by the Taq polymerase, even if the dimer complex is unstable, leading to competition for PCR reagents, and potentially inhibiting amplification of the target DNA sequence. E.g. the 3’ ends of the following hypothetical primer pair are complementary:
· Forward primer: 5’-TGG-CTA-ATT-ATG-3’
· Reverse primer: 5’-GAC-TTG-ACC-CAT-3’
· 5’-TGG-CTA-ATT-ATG-3’ > extension and formation of a primer dimer
· < 3’-TAC-CCA-GTT-CAG-5’
The sequence of the last three nucleotides of the primer (at the 3’-end) should not be complementary to any triplet in either primer. Check this for the 3’end of both primers. Avoid situations as shown below, where the ATG end of the upstream primer is complementary to the 3’-TAC triplet downstream and to 5’-CAT upstream, while the 3’-GTT end of the downstream primer is complementary to the 5’-CAA upstream:
Upstream primer
’--CAA-CAT-ATG--3’
’--CAA-CAT-ATG------CAA-ATG------3’
’--GTT-GTA-TAC------GTT-TAC------3’
3’--GTT-TAC------5’
Downstream primer
· Primer concentration:
Primer concentrations should be between 0.1 – 0.5 µM and can be as high as 1 µM. Higher primer concentrations may promote mispriming and accumulation of non-specific product. Lower primer concentrations may be exhausted before the reaction is completed, resulting in lower yields of the desired product.
Polymerase enzymes
Thermostable DNA polymerases e.g. Taq polymerase have been isolated and cloned from a number of thermophillic bacteria and are used in PCR as they survive the hot denaturation step. Polymerase enzymes read the DNA template and synthesize DNA.
For most applications Taq polymerase is the enzyme of choice. The “Stoffel” fragment of AmpliTaq is analogous to the Klenow fragment of E. coli DNA polymerase I and lacks the intrinsic 5’ ® 3’ exonuclease activity. It is reported to be useful for multiplex PCR (PCR with different primer pairs) and random amplification of polymorphic DNA (RAPD). There are many polymerases depending on their application, commercially available.
Recommended concentration is 1-2.5 Units per 100 ml reaction.
Too high enzyme concentrations result in:
· Non-specific background products
· Decreased specificity (Roche Molecular Biochemical’s. PCR Applications Manual, 1999)
Too low concentrations result in insufficient amounts of product.
Polymerase fidelity is influenced by multiple factors, including the tendency of a polymerase to insert the wrong nucleotide, the presence of a proofreading 3’-5’ exonuclease which can remove mismatches and the ease with which mismatches can be extended.
Magnesium chloride (MgCl2)
Magnesium concentration influences:
· Enzyme activity/fidelity
· Primer annealing
· Strand dissociation temperatures
· Product specificity
· Formation of primer-dimer artifacts
It is therefore important to determine the ideal Mg2+ concentration for each primer pair for a PCR. The optimal MgCl2 concentration may vary from approximately 0.5 mM to 5 mM and can be adjusted for specific reactions.
Deoxynucleotide triphosphate (dNTP)
A generic term referring to the four deoxyribonucleotides:
dATP, dCTP, dGTP, dTTP
dNTPs should be used at equivalent concentrations. Imbalanced dNTPs mixtures will reduce polymerase fidelity. dNTPs reduce free Mg2+, thus interfering with polymerase activity and decreasing primer annealing. A final concentration of between 20-200 mM of each results in an optimal balance in yield, specificity and accuracy.
Thermal Cycling
Initial denaturation (95 °C – 98 °C)
Denaturation is the separation of the DNA double strand into two single strands. It is very important to denature the DNA template completely, and so many thermal cycling programs start with a longer initial denaturation step. If the template DNA is only partially denatured it will tend to “snap-back” very quickly, preventing efficient primer annealing and extension or leading to “self priming” which can lead to false-positive results.
Step 1: Denaturation step during cycling
Denaturation at 95 °C for 20-30 seconds is usually sufficient but must be adapted for the tubes and thermocycler being used.
Step 2: Annealing (45 °C – 65 °C)
The temperature is reduced to allow the primers to anneal. The choice of primer annealing temperature is the most critical factor in designing a high specificity PCR. If the temperature is too high, no annealing occurs. If the temperature is too low, non-specific annealing will increase dramatically. The actual annealing temperature depends on the primer lengths and sequences. After annealing, the temperature is increased to 72 °C for optimal polymerization, which uses up dNTPs in the reaction mix and requires Mg2+.
Step 3: Primer extension (72 °C)
Time depends upon the length and the concentration of the target sequence and upon the temperature. The rate of incorporation varies between 35-100 nucleotides/sec. A 20 second extension is sufficient for fragments shorter than 500 bp and a 40 second extension is sufficient for fragments up to 1.2 kb.
Final extension
After the last cycle the reaction tubes are held at 72 °C for 5-15 minutes to promote completion of partial extension products and annealing of single-stranded complementary products.
Cycle number
Most PCRs include only 25 to 35 cycles. As the cycle number increases non-specific products can accumulate. Actual yield is less than the theoretical maximum.