Basic Polymerase Chain Reaction PCR, Polymerization, Nucleotide, DNA Polymerase, Deoxyribo - Oligonucleotide, Triphosphate, Nucleoside

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Home >> Biotechnology and Genomics >> Polymerase Chain Reaction-PCR and Gene Amplification >>Basic Polymerase Chain Reaction PCR

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Basic PCR

Inorder to understand PCR, the readers should be familiar with the mechanism of DNA replication within the cell. DNA replication involves polymerization of nucleotides using a template DNA strand with the help of an enzyme DNA polymerase, but this reaction invariably requires a primer strand to which further nucleotides can be added using the DNA polymerase enzyme.

If the primer strand is not available, the reaction can not proceed. In the living cells this primer strand is not a DNA strand but is a - small single stranded RNA molecule synthesized with the help of RNA polymerase enzyme, which does not need a primer

A Thermal Cycler for PCR Amplification

A Thermal Cycler for PCR Amplification(the Instrument has a Microprocessor-controlled Temperature Cycling needed for PCR

In PCR, a similar reaction takes place in an 'eppendorf tube', where the primer strand is added from outside in the form ofa deoxyribo-oligonucleotide, and - DNA polymerase enzyme is added to help in polymerization. Unlimited supply of amplified DNA is obtained by repeating the reaction, which is made possible by regular denaturation of freshly synthesized double stranded DNA molecules by heating it to 90-980C. At this high temperature the two strands separate. Once the double stranded DNA is made single stranded by heating up to 90-98'C, the mixture of these two strands with two primers that recognize the sequences bordering the sequence to be amplified is cooled to 40-600C. This allows the primers (which are in excess) to bind to their complementary strands through renaturation.

The expression profile with first generation. Ad vectors is not suitable for the long-term correction of chronic diseases but is adequate for direct cell killing, for most immunotherapy strategies and for some acute diseases. The first generation vectors have an insert –size limit of ~7.5kb. The promoter used most frequently with Ad (and indeed all other vectors) is derived from cytomegalovirus(CMV), which gives strong expression in many cell types.

Ad gives particularly, efficient gene transfer to the liver, such that dissemination form the site of local injection(such as tumours) and consequent liver transfection is the most serious safety concern. The most serious limitation of Ad vectors stems from their tendency to elicit strong immune and (at high doses) inflammatory responses. Single, large doses of Ad provoke neutralizing antibody response directed to proteins of the viral particle, which prevent binding to target cells and abrogate gene transfer upon repeat dosing by systemic administration routes in animals.

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The presence ofTaq DNA polymerase enzyme and all the four essential nucleoside triphosphates allow synthesis of complementary strands in the usual manner. In a thermal cycler, this process is automatically repeated 20-30 times (as predetermined by a computer device), so that in a single afternoon, a billion copies of the sequence flanked by the left and right primers can be produced. Inorder to continue the synthesis, the temperature of the mixture is alternately increased (for denaturation) and decreased (for renaturation) once every 1-3 minutes (as fixed by the computer device).

This requires that during temperature rise, the enzymatic activity of DNA polymerase should not be destroyed, otherwise one may have to add a fresh aliquot of enzyme in each cycle of amplification. This became possible only by the discovery of thermostable enzyme Taq DNA polymerase, isolated from Thermus aquaticus growing in hot springs. This enzyme acts best at 72.C and. the denaturation temperature of 90'C does not destroy its enzymatic activity.

Basic Reaction of the PCR

Basic Reaction of the PCR(only three cycles of PCR are shown; in each cycle primers are shown by solid boxes, template strands are shown by borken lines1

Basic Reaction of the PCR(only three cycles of PCR are shown; in each cycle primers are shown by solid boxes, template strands are shown by borken lines2

Later, other thermostable enzymes like Pflu DNA polymerase isolated from Pyrococcus furiosus and Vent polymerase isolated from Thermococcus litoralis, were discovered and were found more efficient. These enzymes allowed automation of the entire process and automatic PCR thermal cyclers are now avialable (each for a price of around Rs2-4 lakhs), which can amplify DNA sequences at a fast speed unattended.

As pointed out earlier, PCR may eventually replace gene cloning technique discussed in Chapters 2 and 3. This technology is much easier and requires much smaller quantity (nanogram or ng = 10-9g) of DNA. For cloning, DNA is needed, as the starting material, in microgram (g) quantities. A comparison between PCR and gene cloning.

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