BY DAKSHITA NAITHANI

The polymerase chain reaction (PCR) is an in-vitro (laboratory) technique used to produce huge amounts of DNA.

•             PCR is a cell-free amplification method that produces billions of identical copies of any DNA of interest. PCR, which was invented by Karry Mullis in 1984, is today regarded as a fundamental technique for molecular methods. It is the most widely used approach for multiplication of target nucleic acids.

•              The method usually combines complementary nucleic acid hybridization and nucleic acid replication principles, which are applied repeatedly over many cycles to amplify a single and original copy of a nucleic acid target, which is often undetectable by standard hybridization methods, and multiply to 107 or more copies in a short amount of time. In result, it gives a large number of targets which may be identified using a variety of ways.

ADVANTAGES:

•             Despite being simple it is a very powerful technique.

 •            It enables for massive amplification of any particular sequence of DNA given that short sequences on each side of it are known.

•             Improves sensitivity and specificity while allowing for speedier diagnosis and recognition.

PRINCIPLE OF PCR:

Double-stranded DNA in question is denatured, resulting in two independent strands and  each strand is allowed to hybridise using a primer (renaturation). The enzyme DNA polymerase is used to synthesise DNA from the primer-template duplex. To create various forms of target DNA, the three processes of denaturation, renaturation, and synthesis are performed numerous times.

ESSENTIAL REQUIREMENTS FOR PCR:

•             A target DNA which is around 100-35,000 bp in length.

•             Two primers (synthetic oligonucleotides of 17-25 nucleotides length ) that are complementary to regions flanking the target DNA.

•             Four deoxyriobonucleotides  are used(d ATP, d CTP, d GTP, d TTP)

•             MgCl2 (Magnesium Chloride)

•             Nuclease free water

•             Taq DNA polymerase buffer

•             A thermo-stable DNA polymerase is one that can tolerate temperatures up to 95 degrees Celsius.

The target DNA, two primers (in excess), a thermo-stable DNA polymerase (Taq DNA polymerase), and four deoxyribonucleotides are all included in the reaction mixture. It is a method that includes a series of cycles for DNA amplification.

KEY FACTORS OPTIMAL FOR PCR:

•             PRIMERS:

When it comes to determining PCR, these are crucial. Primers with no secondary structure and no complementarity amongst themselves (17-30 nucleotides) are excellent. In PCR, complementary primers can combine to produce a primer dimer, which can be amplified. The replication of target DNA is prevented as a result of this action.

•             DNA POLYMERASE:

Because it can resist high temperatures, Taq DNA polymerase is chosen. After the heat denaturation stage of the first cycle, DNA polymerase is introduced in the hot start procedure. This prevents the misaligned primers from extending, which is common at low temperatures.

Verification or proof reading of exonuclease (3′-5′) activity is absent in Taq polymerase, which might lead to mistakes in PCR products. Tma DNA polymerase from Thermotogamaritama and Pfu DNA polymerase from Pyrococcusfuriosus are examples of thermostable DNA polymerases with proof reading activity.

•             TARGET DNA:

In general, the smaller the target DNA sequence, the greater the PCR efficiency. A mplification of DNA fragments up to 10 kb has been documented in recent years. In PCR, the sequence of the target DNA is also crucial. As a result, CC-rich strand sections obstruct PCR.

•             PROMOTERS AND INHIBITORS:

 Addition of Bovine serum albumin (BSA) improve PCR by shielding DNA polymerase, humic acids which are commonly present in ancient samples of target DNA, hinder PCR.

EACH CYCLE HAS THREE STAGES:

1.            DENATURATION:

The DNA is denatured and the two strands split when the temperature is raised to around 95 degree celsius for about one minute.

2.            RENATURATION OR ANNEALING:

The primers base pair with the complementary regions flanking target DNA strands as the temperature of the mixture is gradually lowered to around 55 degree celsius.  Annealing seems to be the term for this procedure. Due to the high concentration of primer, annealing occurs between each DNA strand and the primer rather than between the two strands.

3.            EXTENSION OR SYNTHESIS:

The 3′-hydroxyl end of each primer is where DNA synthesis begins. By connecting the nucleotides that are complementary to DNA strands, the primers are expanded. The PCR synthesis process is quite similar to the leading strand DNA replication process.  The optimal temperature for Taq DNA polymerase is about 75 degree celsius. (For E.Coli DNA Polymerase is used). By increasing the temperature, the process can be halted (about 95 degree celsius).

Each cycle lasts around 3-5 minutes and in most cases, it is performed on computerised equipment. The corresponding sequence of the second primer lies beyond the new DNA strand linked to each primer. Long templates allude to these additional strands, which will be utilised in the second cycle.

The strands are denatured, annealed with primers, and exposed to DNA synthesis in the second cycle of PCR. Long and short templates are produced at the end of the second round.

The original DNA strands, as well as the short and long templates, are the starting materials for the third cycle of PCR. For each cycle, the procedures are used again and again. About a million-fold target DNA is produced by the conclusion of the 32nd cycle of PCR, according to estimates. As double-stranded molecules build, the small templates containing precisely the target DNA increase.

TYPES OF PCR:

1.            Real-time PCR

2.            Quantitative real time PCR (Q-RT PCR)

3.            Reverse Transcriptase PCR (RT-PCR)

4.            Multiplex PCR

5.            Nested PCR

6.            Long-range PCR

7.            Single-cell PCR

8.            Fast-cycling PCR

9.            Methylation-specific PCR (MSP)

10.          Hot start PCR

11.          High-fidelity PCR

12.          In situ PCR

13.          Variable Number of Tandem Repeats (VNTR) PCR

14.          Asymmetric PCR

15.          Repetitive sequence-based PCR

16.          Overlap extension PCR

17.          Assemble PCR

18.          Intersequence-specific PCR(ISSR)

19.          Ligation-mediated PCR

20.          Methylation –specifin PCR

21.          Miniprimer PCR

22.          Solid phase PCR

23.          Touch down PCR, etc

APPLICATIONS OF PCR:

1.            PCR IN CLINICAL DIAGNOSIS:

PCR’s specificity and sensitivity make it ideal for diagnosing a variety of human illnesses. RFLP is not involved in the development of many genetic diseases (restriction fragment length poly-morphism). For all of these problems, PCR is a godsend since it delivers straight DNA information it is accomplished by amplifying DNA from the appropriate area and then analysing the PCR results directly.

o             PRENATAL DIAGNOSIS OF INHERITED DISEASES:

It is used to diagnose hereditary disorders in the womb utilising chorionic villus samples or amniocentesis cells various c onditions such as sickle cell anaemia, p-thalassemia, and phenylketonuria can thus be identified in these specimens using PCR.

o             DIAGNOSIS OF RETROVIRAL INFECTIONS:

                PCR from cDNA is a useful technique for detecting and maintaining retroviral infections, such as HIV.

o             DIAGNOSIS OF BACTERIAL INFECTIONS:

o             PCR is used for the detection of bacterial infections such as tuberculosis which is caused by Mycobacterium tuberculosis.

o             DIAGNOSIS OF CANCERS:

PCR can identify some virally-induced malignancies, such as cervical cancer caused by the human papillomavirus it can also identify malignancies caused by chromosomal translocations (chromosome 14 and 18 in follicular lymphoma) containing known genes.

o             PCR IN SEX DETERMINATION OF EMBROYS:

The sex of human and animal eggs fertilised in vitro may be identified using PCR using sex chromosome-specific primers and DNA probes. This method can also be used to identify sex-related abnormalities in fertilised eggs.

2.            PCR IN DNA SEQUENCING:

 The process is useful for sequencing since it is considerably easier and faster to amplify DNA. Single strands of DNA are required for this function. Asymmetric PCR involves preferred amplification of a single strand. Strand removal can also be accomplished by digesting one strand.

3.            PCR IN FORENSIC MEDICINE:

For amplification, a single molecule from any source (blood strains, hair, semen, etc.) of a person is sufficient. As a result, PCR is critical for crime detection.

4.            PCR IN COMPARISON WITH GENE CLONING:

In comparison to traditional gene cloning procedures, PCR offers a variety of benefits. Improved efficiency, small amounts of beginning material (DNA), cost-effectiveness, low technical expertise, and the time frame are only a few of them. In the long run, PCR may be able to replace most gene cloning applications.

5.            PCR IN GENE MANIPULATION AND EXPRESSION STUDIES:

The benefit of PCR is that the primers do not need to be complementary to the target DNA. As a result, it may alter and amplify the nucleotide sequence in a portion of the gene (target DNA). The coding sequence of a protein of interest can be changed using this approach. Gene manipulations are also crucial for studying the impact of factors on gene expression.

The study of mRNAs, which are the results of gene expression, requires the use of PCR. Reverse transcription-PCR is used to accomplish this.

6.            PCR IN COMPARITIVE STUDIES OF GENOMES:

PCR using random primers can be used to assess the differences in the genomes of two species. Electrophoresis is used for separation of products for their comparative identification and it is predicted that two genomes from closely related species will produce more comparable bands.

The study of evolutionary biology, more especially phylogenetic biology, relies heavily on PCR. It has transformed palaeontology and archaeological research since it can amplify even minute amounts of DNA from any source (hair, mummified tissues, bone, or any fossilised material).