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PCR
- 2. Polymerase technique chain used reaction (PCR) isa in molecular biology to amplify a single copy or a few copies of a segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.
- 3. Developed in1983 by Kary Mullis, PCR is now a common technique used in clinical and research laboratories for a broad variety of applications. In 1993, Mullis was awarded the Nobel Prize in Chemistry for his work on PCR.
- 4. DNA template istarget DNA sequence. It is the DNA molecule that contains the region (segment) to be amplified, the segment we are concerned which is the target sequence.
- 5. DNA polymerase: DNA polymerasesequentially adds nucleotides complimentary to template strand at 3’-OH of the bound primers and synthesizes new strands of DNA complementary to the target sequence. The most commonly used DNA polymerase is Taq DNA polymerase (from Thermus a thermophillic bacterium)aquaticus, because of high temperature stability.
- 6. DNA polymerasePfu (fromPyrococcus furiosus)is also used widely because of its higher fidelity (accuracy of adding complimentary nucleotide). Mg2+ ions in the buffer act as co-factor for DNA polymerase enzyme and hence are required for the reaction.
- 7. Primers are syntheticDNA strands of about 18 to 25 nucleotides complementary to 3’end of the template strand. DNA polymerase starts synthesizing new DNA from the 3’ end of the primer .
- 8. Two primers mustbe designed for PCR; the forward primer and the reverse primer.
- 9. The forward primerattaches to the start codon of the template DNA (the anti- sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand). The 5' ends of both primers bind to the 3' end of each DNA strand.
- 10. Primers should bindto template with good specificity and strength. If primers do not bind to correct template, wrong sequence sequences and appropriate will get amplified. Optimal primer primer concentrations are essential for maximal specificity and efficiency in PCR.
- 11. Complementary nucleotide sequenceswithin a primer and between primers should be avoided. If there are complimentary sequences in two primers used (one primer for each will hybridize DNA strand), the primers with each other thus forming primer-dimmers and will not be available for binding with template. If there are complementary sequences within a primer, it will make hairpin loop structures as shown below.
- 14. The primersshould preferably end on a Guanine and Cytosine (GC) sequence so that it can attach with sufficient strength with template. This increases efficiency of priming due to stronger bonding of G and C bases.
- 15. All types ofnucleotides are "building blocks" fornew DNA strands and essential for reaction. It includes Adenine(A), Guanine(G), Cytosine(C), Thymine(T) or Uracil(U).
- 16. Magnesium affects primerannealing and template denaturation, as well as enzyme activity. An excess of magnesium gives non-specific amplification products, while low magnesium yields lesser amount of desired product.
- 17. There are threemajor steps in a PCR, which are repeated for 30 or 40 cycles. This is done on an automated cycler, which can heat and cool the tubes with the reaction mixture in a very short time.
- 18. • During theheating step (denaturation), the reaction mixture is heated to 94°C for1min, which causes separation of DNA double stranded. Now, each strand acts as template for synthesis of complimentary strand.
- 20. This step consistof cooling of reaction mixture after denaturation step to 54°C, which causes hybridization (annealing) of primers to separated strand of DNA (template). The length and GC- content (guanine-cytosine content) of the primer should be sufficient for stable binding with template.
- 21. Guanine pairs hydrogen bondingadenine binds with cytosine with three with thymine with two hydrogen bonds. Thus, higher GC content results in stronger binding.
- 23. • The reactionmixture is heated to 72°C which is the ideal working temperature for the Taq polymerase. The polymerase adds nucleotide (dNTP's) complimentary to template on 3’–OH of primers thereby extending the new strand.
- 25. First three stepsare repeated 35-40 times to produce millions of exact copies of the target DNA. Once several cycles are completed, during the hold step, 4–15 °C temperature is maintained for short-term storage of the amplified DNA sample.
- 26. • As bothstrands are copied during PCR, there is an exponential increase of the number of copies of the gene. Suppose there is only one copy of the desired gene before the PCR starts, after one cycle of PCR, there will be 2 copies, after two cycles of PCR, there will be 4 copies. Afterthree cycles there will be 8 copies and so on.
- 31. Polymerase Chain Reaction 1.Diagnosis of bacterial and viral diseases In early phases of tuberculosis, the sputum may contain only very few tubercle bacilli, so that usual acid fast staining may be negative. But PCR could detect even one bacillus present in the specimen. Any other bacterial infection could also be detected. The specific nucleotide sequences of the bacilli are amplified by PCR and then detected by Southern blot analysis.
- 32. 2. Medicolegal cases PCRallows the DNA in a single cell or in a hair follicle to be analysed. The restriction analysis of DNA from the hair follicle from the crime scene is studied after PCR amplification. This pattern is then compared with the restriction analysis of DNA samples obtained from various suspects. The culprit's sample will perfectly match with that of PCR amplified sample.
- 33. 3. Diagnosis ofgenetic disorders The PCR technology has been widely used to amplify the gene segments that contain known mutations for diagnosis of inherited diseases such as sickle cell anemia, thalassemia, cystic fibrosis, etc. 4. Prenatal diagnosis of inherited diseases 5. Cancer detection: PCR is widely used to monitor residual abnormal cells present in treated patients. Similarly, identification of mutations in oncosuppressor genes such as p53, or retinoblastoma gene can help to identify individuals at high risk of cancer.
- 35. Reverse Transcriptase PCR(RTPCR) Instead of Taq polymerase, Tth polymerase from Thermus Thermophilus may be used. This enzyme has both DNA polymerase and reverse transcriptase activities at 95°C. So mRNA is copied to cDNA synthesis followed by PCR amplification. In ordinary PCR, DNA is detected; that DNA could be from a living or non-living organism. But in reverse PCR, mRNA is detected; that means, it is derived from a living organism.
- 36. Reverse Transcriptase PCR(RTPCR) By this method, quantitation of the number of virus present in a sample can be calculated., e.g.,viral load in HIV (human Immuno deficiency virus or HBV (hepatitis B virus).
- 37. A real-time polymerasechain reaction (real-time PCR), also known as quantitative polymerase chain reaction (qPCR), 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), not at its end, as in conventional PCR.
- 38. Real-time PCR iscarried out in a thermal cycler with the capacity to detect the fluorescence emitted by the dye applied. The PCR process generally consists of a series of temperature changes that are repeated 25–50 times. These cycles normally consist of three stages: the first, at around 95 °C, allows the separation of the nucleic acid's double chain the second, at a temperature of around 50–60 °C, allows the binding of the primers with the DNA template the third, at between 68–72 °C, facilitates the polymerization carried out by the DNA polymerase.