Polymerase Chain Reaction, PCR-139, Definition, Principle, Types and application of PCR

Polymerase Chain Reaction, PCR-139
Definition, Principle, Types and application of PCR

1. Polymerase chain reaction By S.D.Mankar Assistant Professor Department Of Pharmaceutics Pravara Rural College Of Pharmacy,Pravaranagar S.D.Mankar 1

2. PCR PCR is labrotory (In Vitro) technique for generation large quantities of specified DNA. Cell- free amplification technique for synthesizing multiple identical copies of any DNA of interest.

3. Principle The double stranded DNA of interest is denaturized to separate into two individual strands. Each strand is then allowed to hybridized with a primer( renaturation ). The primer template duplex is used for DNA synthesis. These three steps denaturation , renaturation & synthesis are repeated again and again to generate multiple forms of target DNA.

4. Gene/DNA cloning Cloning(Amplification) is a process of making identical copies of biological material. A clone is an exact copy of an organism, organ, single cell, gene or macromolecule. Gene cloning is the act of making multiple copies of a single gene. Techniques for gene cloning enable scientists to prepare multiple identical copies of recombinant DNA(gene-sized pieces of DNA). Gene cloning methods: 1. In vivo method 2. In vitro method: PCR. 1. In vivo method: it involves all the fundamental steps that we saw in recombinant DNA technology.

5. Fundamental steps if in vivo gene cloning: Identification and isolation of the desired gene or DNA fragment to be cloned. Insertion of the isolated gene in a suitable vector. Introduction of this vector into a suitable organism/cell called host or expression vector. The vector multiplies within the host cell, producing numerous identical copies not only of itself but also of the gene that it carries. During the division of the host cell, copies of the recombinant DNA molecules are passed on to its descendants and further vector replication takes place. After a large number of cell divisions, a colony of identical host cells is produced. Each cell in the colony contains one or more copies of the clone/ recombinant DNA molecule.

6. 2. In vitro method of gene/DNA cloning- PCR PCR- Polymerase chain reaction: is a in vitro technique for generating large quantities of specified DNA/gene. It is a cell free amplification technique for synthesizing billions of copies of any DNA. Principle of PCR: it involves three basic steps- 1.Denaturation: the double stranded DNA of interest is denatured to separate into two individual strands at 95 C. 2.Renaturation: each strand is then allowed to hybridize with a primer at 40-65 C. 3.Synthesis: DNA Polymerase extends the DNA chain by adding nucleotides to the 3’ ends of the primers at 72 C. These three steps are repeated again & again(20-40 times), doubling the number of DNA copies each time to generate multiple forms of target DNA/gene.

7. Essential requirements for PCR: 1. A target DNA(100-35000 bp in length) 2. Two primers(synthetic oilgonucleotides of 17-30 nucleotides in length). 3. Four deoxyribonucleotides ( dATP - Adenosine triphosphate - Adenine (A), dCTP - Cytosine (C), dGTP - Guanine (G), , dTTP Thymine (T). 4. A DNA polymerase that can withstand at a temperature of 95 C The reaction mixture contains the target DNA, two primers(in excess), a thermostable DNA polymerase(isolated from the bacterium Thermus aquaticus called as Taq DNA polymerase ) & four deoxyribonucleotides . The actual technique of PCR involves repeated cycles for amplification of target DNA. Each cycle has three stages.

8. Each cycle has three stages: 1. Denaturation: on increasing the temperature to about 95 C for about one minute, the DNA gets denatured & two strands separate. High temperature causes the hydrogen bonds between the two strands of DNA to break. This usually takes between 15-30 seconds. 2. Renaturation or annealing: During this stage the reaction mixture is cooled to 50-65 C. This enables the primers to attach to a specific location on the single-stranded template DNA by hydrogen bonding. High concentration of primer ensures annealing between each DNA strand & the primer rather than two strands of DNA. Primers serve as the starting point for DNA synthesis. Because the polymerase enzyme can only add DNA bases to a double strand of DNA.

9. 3. Synthesis: Once the primer has bound,only then the polymerase enzyme can attach and start making the new complementary strand of DNA from the loose DNA bases(A, T, G, C). Thus the primers are extended by joining the bases complementary to DNA strands. The synthetic process in PCR is quite comparable to DNA replication. The optimum temperature for Taq DNA polymerase is around 72 C( for E. coli DNA polymerase is 37 C). The reaction can be stopped by raising the temperature to about 95 C. This is one cycle of PCR. Each cycle requires 3-5 minutes. For the second cycle of PCR, the DNA strands(original+ newly synthesized) are denatured, annealed with primers & subjected to DNA synthesis.

10. Types of PCR 1. Reverse transcription-PCR(RT-PCR): it is used for amplification of RNA molecules. For this, the RNA template is first converted into a complementary DNA (cDNA) using a reverse transcriptase enzyme. The cDNA is then used as a template for exponential amplification using PCR. RT PCR procedure constitutes two steps- First Strand Reaction- RNA strand i.e., mRNA strand is first reverse transcribed into ss (single stranded) cDNA template using reverse transcriptase , through the process of reverse transcription. Second Strand Reaction- After the cDNA is generated, standard PCR is initiated using DNA polymerase. After ~35 cycles, millions of copies of the sequence of interest are generated. The original RNA template is degraded by Rnase leaving pure cDNA.

11. Uses: 1. It is primarily used to measure the amount of a specific RNA for the detection of RNA viruses(Retroviruses) like HIV, COVID 19. 2-Detection of other MOs through targeting of their Ribosomal RNA.

12. One step & two step RT-PCR: The quantification of mRNA using RT-PCR can be achieved as either a one-step or a two-step reaction. The difference between the two approaches lies in the number of tubes used when performing the procedure. In the one-step approach, the entire reaction from cDNA synthesis to PCR amplification occurs in a single tube. However, the starting RNA templates are prone to degradation in the one-step approach, and the use of this approach is not recommended when repeated assays from the same sample is required. In two-step reaction requires that the reverse transcriptase reaction and PCR amplification be performed in separate tubes.

13. 2. Nested PCR: When target DNA & related(contaminating) DNA are sequentially similar then primers may bind to both the DNAs & therefore undesired DNA is also get amplified. However in Nested PCR, specific primers called Nested primers that bind specifically to target DNA are used, which selectively amplifies the target DNA. In the first cycle of PCR, the products are both from target DNA & undesired DNA. But in the second cycle, nested primers are used, that bind selectively to target DNA & amplification proceeds. 3. Real time quantitative PCR: it involves quantification of the PCR products by using a fluorescent compound like eithidium bromide. The principle is that the double stranded DNA molecule bind to eithidium bromide which emits fluorescence that can be detected & DNA can be quantified

14. 4. Asymmetric PCR: it is used for the synthesis of single-stranded DNA molecules. In Asymmetric PCR, two primers in a ratio 100:1 are used. After 20-25 cycles of PCR, one primer is exhausted, as a result in next 5-10 PCR cycles, only single-stranded DNA molecules are generated.

15. Applications of PCR 1. Diagnosis of various diseases: The specificity & sensitivity of PCR is highly useful for the diagnosis of various diseases. Infection with viruses or microorganisms (bacteria, parasites, etc.) results in the presence of their genetic material in all or part of the infected organism. PCR is effective in detecting the presence of a pathogen in a biological(blood, sputum) sample. The performance of the PCR diagnosis is based on a criterion: the choice of primers capable of very selectively amplifying a sequence of the DNA of the virus or microorganism. Prenatal diagnosis of inherited diseases: PCR is used in the prenatal diagnosis of inherited diseases like sickle cell anemia, thalassemia using tissue samples from the chorionic villus (the membranes found between the mother and unborn baby) or foetal tissue from the amniotic fluid (the fluid around the unborn baby).

16. Diagnosis of retroviral infections: PCR is a valuable tool for diagnosis & monitoring of retroviral diseases. It is primarily used to measure the amount of a specific RNA for the detection of RNA viruses(Retroviruses) like HIV, COVID 19 in a biological sample. Diagnosis of bacterial infections : Mycobacterium tuberculosis. Diagnosis of cancer: several viral induced cancers such as cervical cancer caused by human papilloma virus can be detected by PCR. Sex determination of embryos: sex of human & live stock embryos fertilized in vitro , can be determined by PCR, by using primers & DNA probes specific for sex chromosomes. Further this technique is also useful to detect sex- linked disorders in fertilized embryos.

17. 2 . PCR in identification: PCR is remarkably effective at identifying species, varieties, or individuals by genetic fingerprinting. This application is based on the knowledge acquired on genome structure. It is used to amplify nucleotide sequences that are specific to species, variety, or individual. 3. PCR in diagnosis of genetic diseases: PCR is widely used in the detection of genetic diseases. The amplification of all or part of a gene responsible for a genetic disease makes it possible to reveal the deleterious mutations (s), their positions, their sizes, and their natures.

18. 4. PCR in forensic science: One of the most famous uses for PCR is in the creation of a genetic fingerprint (also known as DNA profiling) from a sample of blood or semen, or from a hair root. Thus PCR is very important for identification of criminals. PCR also plays a role in mitochondrial DNA analysis, used for samples from hair shafts and bones when other samples are not available. With the advent of PCR-based DNA fingerprinting, PCR became an invaluable tool in forensic investigations. Using DNA fingerprinting, tiny fragments of DNA can be isolated from a crime scene and compared to a huge database of DNA of convicts or criminals. It is also useful in ruling out suspects as part of an investigation.

19. 5. Specialized applications of PCR in genetic research: Study of gene expression patterns is another common application of PCR, where cells or tissues are analyzed in different stages to check for expression of a specific gene. qPCR can be used here to quantitate the level of gene expression. PCR also assists techniques like DNA sequencing using which segments of DNA from an area of interest can be easily amplified to study genetic mutations and their consequences. Advanced variants of the PCR technique have been found to be useful in chromosomal analysis techniques that can help in early detection of genetic birth defects in children. PCR supports the traditional method of DNA cloning by amplifying tiny DNA segments for introduction into a vector.

20. 6.DNA sequencing: PCR much simpler & quicker to amplify the DNA- used for DNA sequencing. Single strand of DNA is used.