BASIC STEPS OF PCR
EXPONENTIAL PHASE: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise.
LINEAR(HIGH VARIABILITY): The reaction components are being consumed, the reaction is slowing, and products are starting to degrade.
PLATEAU (End point detection: Gel detection for traditional methods): The reaction has stopped, no more products are being made and if left long enough, the PCR products will begin to degrade.
POLYMERASE CHAIN REACTION
The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It was developed by Kary Mullis in 1983. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.
PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.
Components and reagents of PCR:
DNA template- That contains the DNA region (target) to be amplified.
Primers- Which are complementary to the DNA regions at the 5' (five prime) or 3' (three prime) ends of the DNA.
DNA polymerase, such as Taq polymerase (Layer et al., 1976) or another DNA polymerase with a temperature optimum at around 70°C.
Deoxynucleoside triphosphates (dNTPs)- Also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.
Buffer solution- Providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.
Divalent cations- Magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis
Monovalent cation- Potassium ions.
Initialization step- This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes.
Denaturation step-This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.
Annealing step- The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.
Extension/elongation step- The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C, (Lawyer FC et.al 1993) and commonly a temperature of 72°C is used with this enzyme.
Final elongation- This single step is occasionally performed at a temperature of70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.
Final hold- This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.
The PCR is commonly carried out in a reaction volume of 20-150 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler for about 20 t0 40 cycles.
A primer is a short synthetic oligonucleotide which is used in many molecular techniques from PCR to DNA sequencing. These primers are designed to have a sequence which is the reverse complement of a region of template or target DNA to which we wish the primer to anneal..
When designing primers for PCR, sequencing or mutagenesis it is often necessary to make predictions about these primers, for example melting temperature (Tm) and propensity to form dimers with itself or other primers in the reaction. The following program will perform these calculations on any primer sequence or pair.
Considerations for designing primers-
1. Primers should be 17-28 bases in length
2. Base composition should be 50-60% (G+C)
3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming
4. Tms between 55-80oC are preferred
5. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product
6. Primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided
7. Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided.
Optimization of PCR reaction components:
Initially, there was some variation from test to test when the same PCR program was used. Solving this reproducibility problem required adjustments of PCR components.
Amount of primer- Initially equimolar primer concentrations of each can be used in the multiplex PCR, if there was uneven amplification, with some of the products barely visible even after the reaction was optimized for the cycling conditions, changing the proportions of various primers in the reaction, with an increase in the amount of primers for the "weak" amplicons and a decrease in the amount for the strong"amplicons.
MgCl2 concentrations- A recommended magnesium chloride concentration in a standard PCR is 1.5 mM at dNTP concentrations of around 200 mM each. If the amplification was not complete for all the primers varying concentrations of MgCl2 ranging from 1.5mM to 3mM concentrations.
Amount of template DNA- Recommended Template DNA concentrations for Normal PCR is 10 ng to 50 ng. For a multiplex PCR depending on the intensity of the amplicons template concentrations can vary between 50 ng to 200ng.
Amount of Taq DNA polymerase- Different concentrations of Taq DNA polymerase can be used for performing PCR reaction the optimum concentration per reaction is 0.3 mL or 1U/25 mL reaction volume. Too much. enzyme, possibly because of the high glycerol concentration in the stock solution, can result in an unbalanced amplification.
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