What is PCR?
-DNA Refresher

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PCR Tutorial

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Natural replication of genetic information occurs as a precursor to cell division. When cells divide, DNA from the parent cell is replicated and passed onto two new daughter cells. The resulting daughter cells contain chromosomes that are identical to those within the parent cell. An exception is when cells involved in sexual reproduction are produced eg. sperm and ovum. DNA is the source code for making RNA and proteins to act as enzymes, messengers, transmitters and structural elements of cells.

The genetic material from a single human cell is made up of DNA double strands containing a total of 3 billion pairs of bases. The double strands of DNA are coiled to form what is called the a (alpha) double helix. The double helix is supercoiled, allowing it to fit inside a cell. Stretched end to end the DNA in one human cell would be approximately two metres long. Remarkably, DNA is so twisted and coiled that it fits into a cell nucleus only a thousandth of a millimetre in diameter.

DNA Structure

If the DNA helix is unwound, the structure can be easily recognized as two long strands connected by rungs to resemble a ladder. The uprights of the ladder are made up of sugar and phosphate molecules. The sugar molecule has a pentose (five sided) ring structure, where the 3’ carbon has a hydroxyl group and the 5’ carbon is part of a methyl group. These 3’ and 5’ carbons are the bonding locations for the phosphate groups, which together form the DNA backbone.

The sugar molecules are covalently linked to each other via the phosphate molecules. The sugar-phosphate backbone of each strand is oriented in the opposite direction (antiparallel) to that of the "partner" DNA strand. One runs in the 5’ to 3’ direction and the other runs in the 3’ to 5’ direction.

The covalent bonds between the sugar and phosphate molecules are relatively strong and can withstand heating to 90oC, making them stable during the denaturation step of PCR.

The rungs of the ladder are composed of four types of molecules known as bases. These bases are Adenine, Guanine, Thymine, and Cytosine. Adenine and Guanine are purines ie. one ring structures. Cytosine and Thymine are pyrimidines ie. two ring structures. It is the sequence of these bases which codes for the genetic information.

Purine bases join with pyrimidine bases through hydrogen bonding. The bases in each DNA strand bond in specific complementary pairs, Adenine to Thymine and Guanine to Cytosine, to bases in the facing DNA strand forming the rungs of the genetic ladder. Guanine links with Cytosine via three hydrogen bonds. Thymine link with Adenine via two hydrogen bonds. RNA contains the base Uracil rather than Thymine.

Bases are attached to the DNA backbone by bonding to the sugar molecules, forming two sugar-phosphate backbones linked together via hydrogen bonds.

Highly Conserved Regions of DNA

Highly conserved regions of DNA are specific sequences of base pairs which code for specific proteins that collectively are representative of each organism. The genetic code, or genome, of each organism contains highly conserved coded regions that are common for the type of organism and variable regions that result in individual traits.

Selection of a highly conserved region of DNA, specific for the target organism, during DNA amplification produces the high specificity of the PCR test methodology. The amplification of a specific nucleotide sequence provides enhanced sensitivity for a PCR based test compared to conventional methods.


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