POLYMERASE CHAIN REACTION POLYMERASE CHAIN REACTION AN OVERVIEWAN OVERVIEW

Dr.Anil KumarProfessor & Head

School of BiotechnologyDevi Ahilya University, Khandwa Road

Indore 452 017

Polymerase chain reaction (PCR) is a technique sensitive enough to amplify small DNA fragments a billion folds.

The generation of amplified fragments (amplicons) by conventional PCR or by the modified methods of the technique is extensively used to address a variety of issues related to biology, medicine and forensic sciences.

By PCR, not only the genetic diseases are diagnosed rapidly but ailment caused due to several other reasons such as protozoan, parasites, bacteria or even viruses may be diagnosed and monitored with much ease and accuracy.

LIMITATIONS OF PCRLIMITATIONS OF PCRPCR is a sensitive technique and therefore highly susceptible tocontamination which can result in false positivity.

Various measures are recommended to reduce contamination problems such as:

Single use of reagents Different rooms for DNA specimen preparation, PCR

amplification and post-PCR detection Use of UV or gamma irritation to inactivate or destroy

contaminating DNA. Over and above, a clean work habit ensures trouble free and reliable results.

BASIC PRINCIPLEBASIC PRINCIPLEPCR follows principle of DNA replication…

COMPONENTS OF PCRCOMPONENTS OF PCR Target sequence ( DNA purified by any method viz. alkali lysis, column, glass beads boiling or microwave

treatment) Oligonucleotide primers

• 17-35 mer• GC % -50-60%• Tm -55-65°C• Ta - ~-5°C of Tm• No hairpin, dimer, cross-dimer or false priming• 3’ end specificity• 5’ end stability (GC clamp)

dNTPs Taq DNA polymerase Buffer (50mM KCl, 10mM Tris-Cl(pH 8.3) and 1.5mM

MgCl2

EXPERIMENTAL PARAMETERSEXPERIMENTAL PARAMETERS Initial denaturation: Important for complete unwinding

of genomic DNA. Denaturation: The denaturation and annealing steps following the initial set-up rarely need to be more than 60 sec in duration. 90-95 degrees C for denaturation and Annealing: 50°C for reannealing is recommended . The higher the annealing temperature, the greater the

specificity of hybridization of the oligonucleotide primer. Extension: At 72 °C Final Extention: 72°C for 5 to 10 min

Adding too much polymerase may well increase non-specific amplification

Number of cycles : Between thirty and forty cycles is usually sufficient.

Other consideration :Always carry out PCRs under a layer of sterile mineral oil to prevent evaporations of the reaction solution. If possible, the reaction volume should be less than 100 microlitres to ensure rapid temperature equilibration. If a dry-heat block is used, a little mineral oil added to the wells will ensure good thermal contact.

Samples of the reaction mixture may be withdrawn during the course of a PCR experiment in order to monitor the progress of the amplification.

STANDARD PCR REACTION CONDITIONS (IN 50 MICROLITRES VOLUME)

5X Buffer (containing 67 mM Tris- HCL, pH 8.8 16.6 mM ammonium sulfate, 170 microgram/ml BSA, 6.7 micro

Molar EDTA) 6.7 mM magnesium chloride 10 mM mercaptoethanol (Optional) 200 micro gram/ml dNTP  10 ng linearized DNA 0.3 micro gram primers  0.3 micro gram primers Steile distilled water to make up the volume -

- Use sterile, siliconised 0.5 ml capped microfuge tubes.

VISUALIZING RESULTS:

1.2% agarose gels or 5-10% polyacrylamide are used dependingon the size of the DNA segment being amplified. 

Confirmations

Restriction Digestions Southern blotting followed by hybridization with a suitable

probe may reveal amplified sequences, which are not visible by ethidium bromide staining.

Nested PCR

ELISA can also be used for the confirmation. 

PRECAUTIONS

Precautions should be taken to guard against contamination of the reaction mixture with trace amounts of DNA that could serve as templates.

Assemble and carry out polymerase chain reactions in a laminar flow hood equipped with ultraviolet lights. Turn on these lights whenever the hood is not in use. Keep in the hood a microfuge, disposable gloves, supplies, and sets of pipetting devices used only to handle polymerase chain reactions. Since the barrels of automatic pipetting devices are common sources of contamination, prepare and handle reagents using positive displacement pipettes equipped with disposable tips and plungers. Autoclave all buffers, pipette tips, and centrifuge tubes before use.

MODIFICATIONS OF PCR 

Modified forms of PCR are available which help in the exploitation of this versatile technique in wide number of applications. The main modifications are discussed below.

 AMPLIFICATION REFRACTORY MUTATION SYSTEM

(ARMS) PCR  ARMS is also known as allele-specific PCR (ASPCR) or PCR

amplification of specific alleles (PASA). It was first described by Newton and co-workers in 1989. ARMS PCR is used to detect point mutations and small deletions/insertions in the genomic DNA. It may also be used in HLA (human leukocyte antigen) typing. ARMS PCR can generally detect a single copy of the mutant allele amongst 40 copies of the normal. It generates rapid, reproducible and inexpensive results.

Principle of the technique. A PCR primer is designed so that it can discriminate between templates which differ at a specific single nucleotide residue. It is based on the principle that Taq polymerase has no 3' to 5' exonuclease activity; therefore a mismatch at the 3' end of the primer and the template will result in its inability to function as a primer under appropriate conditions.

A standard ARMS PCR consists of two complementary reactions (two tubes) and utilizes 3 primers. One primer isconstant and complementary to the template in both reactions, the other primers differ at their 3' terminal residues and are specific to either the wild type DNA sequence or the mutated sequence at a given base. Only one of these primers is used per tube. If the sample is of a homozygous mutant or homozygous wild type, amplification will only occur in only one of the tubes, if the sample is heterozygous, amplification will be seen in both the tubes.

 The ARMS primer is designed so that the nucleotide distinguishingbetween the different alleles is at the 3' terminal base of the primer.Ideally a control set of primers should be included in the PCR as an internal amplification control. ARMS Primers are usually ~30 bases long (14-30 have been used). Low dNTPs concentrations result in a more specific ARMS reaction (100 μM). Reducing oligonucleotide (primer) concentrations to 0.05μM may increase specificity.

MODIFICATIONS OF ARMS METHODOLOGY 

MULTIPLEX ARMS PCR. In this method a number of ARMS primers, each specific for a particular allele, are included in a single reaction. PCR products corresponding to different alleles are separated due to physical characteristics viz. length. This modification exempts the need for control primers to be added to the reaction and ARMS reactions act as internal controls for each other. Besides this allows a bi-allelic system to be genotyped in one tube (4 primers: 2 ARMS primers­-allele specific and 2 Non ARMS primers and a control product is obtained from the 2 non ARMS primers).

 DOUBLE ARMS PCR. Two allele specific primers are used

simultaneously in a single reaction (no Non-ARMS primers used in the reaction). Amplification only occurs when the alleles specific for each of the ARMS primers are present on the same chromosome.

Double ARMS PCR is a powerful tool in clarifying haplotypes, particularly in individuals who are doubly heterozygous at two polymorphic sites. Double ARMS PCR is useful in human leukocyte antigen (HLA) typing. Double ARMS PCR has been shown to detect a single target molecule in an excess of 105

background molecules. Hence, this technique is used to detect fetal cells in maternal blood.

 

FLUORESCENT ARMS PCR. The wild type and mutant primers are labeled with different fluorochromes, and are then incorporated into the same reaction tube. This is a more robust test, having much increased specificity. 

REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION (RT-PCR) 

Generally for PCR, DNA is used as the template. However, in a modified method called RT PCR, RNA is used as the template for PCR. This approach involves the use of random primer usually a hexamer or an oligo dT primer in case of RNA. If oligo dT primer is used, since it is complementary to the 3' poly A tail of the mRNA, it anneals to the mRNA used as the target. This form of PCR enables to quantitate mRNA levels and to generate cDNA fragments. The RNA strand is subjected to reverse transcription by the enzyme reverse transcriptase in a PCR reaction. The cDNA synthesized can then be used for PCR amplification using the conventional PCR technique.

RT PCR is particularly useful for studying the expression of genesand for monitoring obscure species of mRNA. It is also used for cloning or subcloning and for the identification of bacterial colonies. 

COMPETITIVE AND QUANTITATIVE RT-PCR.

COMPETITIVE RT-PCR - In this type of PCR there is no real competition in the biochemical sense as all reagents are in excess. The only limiting factor is the copies of cDNA taken in the first few cycles.

QUANTITATIVE RT -PCR - For quantifying mRNA, internal standard RNAs are added in a defined quantity to the RNA sample prior to the RT reaction. The resulting standard cDNA is co -amplified with the same primers as the endogenous target sequence. Its PCR - product is approximately 50 nucleotides smaller. This method allows measurement of small differences in mRNA amount in the RNA samples.

ARBRITARILY PRIMED POLYMERASE CHAIN REACTION (AP-PCR)

 

This type of PCR requires only a single primer of a smaller length as compared to the primers used in conventional PCR. The primeranneals to both the strands arbitarily and fragments of various lengths are randomly generated resulting in a number of bands when observed on the agarose, gel after electrophoresis. The number of bands obtained depends on the primer length, sequence complexity and the available annealing sites complementary to the primer used.

The AP PCR is used as a powerful technique along with DNA fingerprinting to obtain the genetic profile of plants and animals and in forensic science. 

ASYMMETRIC PCR

In this modified approach, target sequence of one strand can beamplified several orders of magnitude as compared to itscomplementary strand. In this type of PCR the quantity of one of theprimer corresponding to one strand is reduced hundred folds. Theprimer for the other strand of interest is present in normal quantity.

As PCR starts, the initial amplification cycles of both the strandsproceeds at the same speed and amplicons are generated from both the strands. However, as the reaction proceeds and the number of DNA strands to be copied increases, the strand which has a reduced quantity of primers lags behind the other since the number of primer molecules present for binding to the large number of newly generated fragments is lesser.

Inverted PCR 

Conventional PCR allows the amplification of a DNA segment flanked by the two primers. But this does not permit the acquisition of sequence information out side the boundary defined by the primers. This limitation can be overcome by Inverted PCR. In this method instead of primers facing each other as is used in conventional PCR, the primers used are such that they face outwards after annealing to the two strands of DNA.

For this, the target DNA is digested with a restriction enzyme or sheared. mechanically to generate linear DNA fragments. This linear DNA is circularized by the action of DNA ligase. The two primers are annealed with the circular DNA, which face each other flanking the unknown sequence. This allows the amplification of the unknown sequence.

Mutagenically separated PCR (MS-PCR) 

In this method given by Rust and co workers in 1993, normal and mutant primers are designed, but they are of different lengths. These two primers are then used in one single reaction with a common other primer. The resultant PCR products differ in size and can be separated on this basis. (1 reaction 3 primers)

Nested PCR 

It involves one PCR reaction followed by the next PCR extension which amplifies the first PCR product. Two sets of primers are designed. PCR is first carried out using outer primers and subsequently with inner primers positioned with in the product obtained in the first extension.. While designing primers.   

For nested PCR, care should be taken to eliminate potential primer dimmers and cross dimmers with in and between inner and outer primer sets. Nested PCR is successful in reducing unwanted products and increases sensitivity. It is generally used when the actual quantity of target DNA is very low or when the target DNA is impure.

APPLICATIONS OF PCR 

Amplification of human specific DNA sequences. Differentiation of species, sub-species and strains.

PCR amplification for DNA sequencing. For detection of mutations. For monitoring cancer therapy. To detect,bacterial and viral infections. For pre determining the sex. For linkage analysis using single sperm cells. For studying Molecular evolution For ascertaining the recombinant clones. and much more.….

 

““Real-Time” or Kinetic PCRReal-Time” or Kinetic PCR

Provides investigators the ability to perform very sensitive, accurate, and reproducible measurement levels of gene expression.

Can be used in measuring viral load, performing allelic discrimination studies, and optimizing PCR conditions.

Real-Time PCR ChemistryReal-Time PCR ChemistryThe 5’ nuclease assay, provides a real-time method for detecting only specific amplification products unlike intercalator-based method which generate signal for both specific and non-specific products.

During amplification, probe annealed to the target sequence is cleaved by the 5’ nuclease activity of Taq polymerase when the enzyme extends from an upstream primer into the region of probe.

Fluorogenic probes made it possible to eliminate post-PCR processing for the analysis of probe degradation.

ProbeProbeProbe is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by Forster resonance energy transfer (FRET) through space.

Probe anneals downstream to the primer sites and is cleaved by the 5’ nuclease activity of Taq DNA polymerase as the primer is extended.

Cleavage separates the reporter dye from the quencher dye, increasing the reporter signal.

Intensity of fluorescence signal increase as additional reporter dye molecules are cleaved from their respective probes with each cycle.

AdvantagesAdvantages

Only specific hybridization between probe and target generate fluorescent signal.

Fluorogenic probes can be labeled with different, distinguishable reporter dyes, thus amplification of two different sequences can be detected in a single PCR reaction.

DisadvantageDifferent Fluorogenic probes must be synthesized to detect different sequences.

Real-Time PCR QuantitationReal-Time PCR Quantitation

Revolutionizes the way one approaches PCR-based quantification of DNA and RNA. Reactions are characterized by the point in time during cycling when amplification of PCR product is first detected rather than the amount of PCR product accumulated after a fixed number of cycles.

Higher the starting copy number of nucleic acid target, the sooner a significant increase in fluorescence is observed.

An amplification plot is a plot of fluorescence signal versus cycle number. There is little change in fluorescence signal in initial cycles of the PCR.

An increase in fluorescence above the baseline indicates the detection of accumulated PCR product.

A fixed fluorescence threshold can be set above the baseline.

The parameter CT (threshold cycle) is defined as the fractional cycle number at which the fluorescence passes the fixed threshold.

Quantification of the amount of target in unknown samples is accomplished by measuring CT and using the standard curve to determine starting copy number.