PCR - Polymerase Chain Reaction

What is PCR ?
Steps in PCR
Applications of PCR

1. POLYMERASE CHAIN REACTION (PCR) Shreyaskumar Jitendra Patel

2. PCR A technique used in molecular biology to amplify a single copy or a few copies of a desired segment of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. It is an easy, cheap, and reliable way to repeatedly replicate a focused segment of DNA, a concept which is applicable to numerous fields in modern biology and related sciences. It was developed in 1983 by Kary Mullis and is now a common and often indispensable technique used in clinical and research laboratories for a broad variety of applications. PCR actually amplifies only a chosen segment (target sequence) within the original DNA template, not the whole template DNA molecule.

3. Components used in PCR 02 03 that is to be copied is called the template, the segment of it that will actually be amplified is known as the target sequence. original DNA molecule needed to initiate DNA synthesis. These are short pieces of single stranded DNA that match the sequences at either end of the target DNA segment Two PCR primers needed to manufacture the DNA copies. The PCR procedure involves several high temperature steps so a heat resistant DNA polymerase is required. enzyme DNA polymerase 01 Thermostable polymerases are isolated from bacteria living in hot springs at temperatures up to 900 C. Taq polymerase from Thermus aquaticus is most widely used as it is stable at high temperatures remaining active even after DNA denaturation, thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

4. Components used in PCR 05 06 A supply of nucleotides is needed by the polymerase to make the new DNA. These are supplied as the nucleotide triphosphates/deoxynucleotide triphosphates (dNTPs). nucleotides needed to keep changing the temperature. The PCR process requires cycling through several different temperatures. Because of this, PCR machines are sometimes called thermocyclers. The reaction is commonly carried out in a volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. PCR machine providing a suitable chemical environment for optimum activity and stability of the DNA polymerase. buffer solution 04

5. Components of PCR

6. CYCLING THROUGH PCR Typically, PCR consists of a series of 20–40 repeated temperature changes, called cycles. There are 3 basic steps in each cycle of PCR: template denaturation, primer annealing elongation of new strands of DNA.

7. Steps in PCR Cycle This step is only required for DNA polymerases that require heat activation by hot-start PCR. It consists of heating the reaction chamber to a temperature of 94–96 °C, or 98 °C if extremely thermostable polymerases are used, which is then held for 1–10 minutes. 1. Initialization : If initialization is not required, the template DNA is denatured by heating to 90 ° C for a minute. This causes DNA melting or denaturation of the double stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single stranded DNA molecules. Although the primers are present from the beginning, they cannot bind to the template DNA at 90 ° C 2. Denaturation :

8. Steps in PCR Cycle The temperature is dropped to around 50-60 ° C for 20–40 seconds allowing the primers to anneal to complementary sequences on the template strand. A longer primer is more specific for binding to the exact target sequence. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3' end of each strand. It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. 3. Annealing :

9. Steps in PCR Cycle The temperature is increased to 700C for a minute or two to allow the thermostable polymerase to elongate new complementary DNA strands starting from the primers. DNA synthesis goes from 5’ to 3’ for both new strands. This gives 2 partly double stranded pieces of DNA. The 2 new strands are not as long as the original templates. They are each missing a piece at the end where synthesis started. However, they are double stranded over the region that matters, the target sequence. The 3 steps are repeated in each cycle of PCR. After repeating the same 3 steps, the second cycle produces 4 partly double stranded pieces of DNA. Although they vary in length, they all include double stranded DNA from the target region. As the cycles continue, the single strand overhangs are rapidly outnumbered by segments of DNA containing only the target sequence. Once past the first two or three cycles, the vast majority of the product is double-stranded target sequence with flush ends. 4. Extension :

10. Steps in PCR Cycle This single step is optional, but is performed at a temperature of 70– 74°C (the temperature range required for optimal activity of most polymerases used in PCR) for 5– 15 minutes after the last PCR cycle to ensure that any remaining single stranded DNA is fully elongated. 5. Final elongation : The final step cools the reaction chamber to 4–15 °C for an indefinite time, and may be employed for short-term storage of the PCR products. 6. Final hold : Finally, the DNA generated is run on an agarose gel to assess the size of the PCR fragment.

11. ADVANTAGES PCR has a number of advantages. It is fairly simple to understand and to use, and produces results rapidly. The technique is highly sensitive with the potential to produce millions to billions of copies of a specific product for sequencing, cloning, and analysis. qRT -PCR shares the same advantages as the PCR, with an added advantage of quantification of the synthesized product. Therefore, it has its uses to analyze alterations of gene expression levels in tumors, microbes, or other disease states DISADVANTAGES One major limitation of PCR is that prior information about the target sequence is necessary in order to generate the primers that will allow its selective amplification. This means that, typically, PCR users must know the precise sequence(s) upstream of the target region on each of the two single stranded templates in order to ensure that the DNA polymerase properly binds to the primer-template hybrids and subsequently generates the entire target region during DNA synthesis. Like all enzymes, DNA polymerases are also prone to error, which in turn causes mutations in the PCR fragments that are generated.

12. A researcher is performing PCR to amplify a sample of DNA. Unfortunately, he forgot to add the DNA primer prior to starting the experiment. Which of the following results is he most likely to observe? A. The reaction will work, but at a significantly slower rate B. The reaction will work, but the product will contain many undesired mutations C. The reaction will be completely unsuccessful D. The reaction will work, but amplify a region that was not his target After four cycles of thermocycling, how many copies of the targeted region will be in the PCR product? A. 2 B. 32 C. 64 D. 16

13. Majority of the time PCR amplifications include an Initialization step, also known as the Hot Start. What is the purpose of the Hot Start? A. Denaturation of the DNA strands B. Activation of the DNA polymerase C. Extension of the template strand D. For primers to begin binding to the template strand Adenylation of the new DNA strands by DNA

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