Variants of Basic PCR method, their principle, purpose and applications

PCR ( PolymeraseChain Reaction)

The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.

Principle of PCR

PCR uses the enzyme DNA polymerase that directs the synthesis of DNA from deoxynucleotide substrates on a single-stranded DNA template. DNA polymerase adds nucleotides to the 3` end of a custom-designed oligonucleotide when it is annealed to a longer template DNA. Thus, if a synthetic oligonucleotide is annealed to a single-stranded template that contains a region complementary to the oligonucleotide, DNA polymerase can use the oligonucleotide as a primer and elongate its 3` end to generate an extended region of double stranded DNA.

           

Applications and purpose

• ·          Selective DNA isolation
• ·        Amplification and quantification of  DNA
• ·        Disease Diagnosis
• ·        Forensic Sciences

Variants of polymerase chain reaction

1. Multiplex-PCR 

Multiplex –PCR uses several pairs of primers annealing to different target sequences. This permits the simultaneous analysis of multiple targets in a single sample. For example, in testing for genetic mutations, six or more amplifications might be combined.

Principle

This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.

Applications and purpose

·         Some of the applications of multiplex PCR include

·         Pathogen Identification

·         High Throughput SNP Genotyping

·         Mutation Analysis

·         Gene Deletion Analysis

·         Template Quantitation

·         Linkage Analysis

·         RNA Detection

·         Forensic Studies

·         Diet Analysis

2.Real time PCR

A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively (Quantitative real-time PCR), semi-quantitatively (Semi quantitative real-time PCR) or qualitatively (Qualitative real-time PCR).

   

Principle

Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase.

Applications and purpose

·    Real time PCR is applied to rapidly detect nucleic acids that are diagnostic of, for example, infectious diseases, cancer and genetic abnormalities

·    Real time PCR is also used by microbiologists working in the fields of food safety, food spoilage and fermentation and for the microbial risk assessment of water quality (drinking and recreational waters) and in public health protection

·        Detection of phytopathogens

·        Detection of genetically modified organisms

3. Hot-start PCR

It is a technique performed manually by heating the reaction components to the DNA melting temperature (e.g. 95 °C) before adding the polymerase. In this way, non-specific amplification at lower temperatures is prevented. Alternatively, specialized reagents inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody, or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. 'Hot-start/cold-finish PCR' is achieved with new hybrid polymerases that are inactive at ambient temperature and are only activated at elevated temperatures.

Principle

 Hot start PCR works by using a more sensitive technique than standard PCR that allows amplification of low-abundance targets and single-copy genes while reducing PCR background problems.

4. Assembly PCR

Assembly PCR is  (also known as Polymerase Cycling Assembly or PCA) is the synthesis of long DNA structures by performing PCR on a pool of long oligonucleotide s with short overlapping segments, to assemble two or more pieces of DNA into one piece. It involves an initial PCR with primers that have an overlap and a second PCR using the products as the template that generates the final full-length product. This technique may substitute for ligation-based assembly.

Principle

Single-stranded oligos or a mix of single- and double-stranded DNA is used to produce longer genes of up to several thousand base pairs. 

Applications and purpose

• ·        Used in novel gene synthesis
• ·        Cloning double stranded DNA into plasmid vector

5. Allele-specific PCR:

A diagnostic or cloning technique based on single-nucleotide variations (SNVs not to be confused with ( SNPs) (single-base differences in a patient). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNV (base pair buffer around SNV usually incorporated.

Principle

Based on a single nucleotide polymorphism  (SNP, s). PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.

Applications and purpose

• ·         Clinical HIV genotyping
• ·         SNP genotyping

6. Suicide PCR

It is typically used in paleogenetics or other studies where avoiding false positives and ensuring the specificity of the amplified fragment is the highest priority. It was originally described in a study to verify the presence of the microbe Yersinia pestis in dental samples obtained from 14th Century graves of people supposedly killed by plague during the medieval Black Death epidemic.

Principle

The method prescribes the use of any primer combination only once in a PCR (hence the term "suicide"), which should never have been used in any positive control PCR reaction, and the primers should always target a genomic region never amplified before in the lab using this or any other set of primers. This ensures that no contaminating DNA from previous PCR reactions is present in the lab, which could otherwise generate false positives.

Applications and purpose

• ·         Used in the field of palaeomicrobiology
• ·         Diagnosis of Rickettsioses

7.Reverse transcription PCR (RT PCR)

In reverse transcription polymerase chain reaction (RT-PCR), first a RNA strand (template) is reverse transcribed into its complementary DNA copy using reverse transcriptase, and subsequently cDNA is amplified using PCR.Various types of Reverse transcriptase enzyme, isolated from Avian myeloblastosis virus (AMV),Moloney murine leukemia virus (MMLV or MuLV) are generally used to produce a DNA copy from RNA template. Random primers, an oligo (dT) primer or sequence-specific primers are used to amplify cDNA.

Principle

In RT-PCR, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase. The cDNA is then used as a template for exponential amplification using PCR.

• ·        RT-PCR can also be very useful in the insertion of eukaryotic genes into      protectorates
• ·        RT-PCR can be used to diagnose genetic disease such as Lesch–Nyhan        syndrome.
•        Cancer Detection