PCR基础知识[PCR basics]

The polymerase chain reaction

PCR[PCR: Polymerase chain reaction. A method for amplifying a DNA sequence in large amounts, using a heat-stable polymerase and suitable primers to direct the amplification of the desired region of DNA.] is a rapid, inexpensive and simple way ofcopying specific DNA[DNA:Deoxyribonucleic acid, a double chain of linked nucleotides (having deoxyribose as the sugar component), which is the fundamental molecule of which genes are composed.]fragments from minute quantities of source DNA material

• It does not necessarily require the use of radioisotopes or toxic chemicals

• It involves preparing the sample DNA and a master mix with primers[Primer: A short DNA or RNA fragment annealed to a singled-stranded DNA and to which further nucleotides can be added by DNA polymerase.], followed by detecting reaction productsThe polymerase[Polymerase: General term for enzymes that carry out the synthesis of nucleic acid, using a pre-existing nucleic acid template and appropriate nucleotides (viz. ribonucleotides for RNA and deoxyribonucleotides for DNA).] chain reaction (PCR; Erlich, 1989) is a powerful technique that has rapidly become one of the most widely used techniques in molecular biology because it is quick, inexpensive and simple. The technique amplifies specific DNA fragments from minute quantities of source DNA material, even when that source DNA is of relatively poor quality.

Reference

Erlich, H.A. 1989. PCR technology: principles and applications for DNA amplifications.Stockton Press, NY.

PCR procedures: steps

! Denaturation[Denaturation: The separation of the two strands of the DNA double helix, or the severe disruption of a complex molecule without breaking the major bonds of its chains.]: DNA fragments are heated at high temperatures, which reduce the DNA double helix [Double helix: The structure of DNA first proposed by Watson and Crick, with two linked helices joined by hydrogen bonds between paired bases.]to single strands.These strands become accessible to primers

! Annealing[Annealing: Spontaneous alignment of two single DNA strands to form a double helix.]: The reaction mixture is cooled down.Primers anneal to the complementary regions in the DNA template [Template: A molecule that serves as the pattern for synthesising another molecule, e.g. a single-stranded DNA molecule can be used as a template to synthesise the complementary nucleotide strand.]strands, and double strands are formed again between primers and complementary sequences[Complementary sequence: The sequence of a DNA or RNA strand to which a given nucleotide sequence can bond to form a double-stranded structure, e.g. TAGGAT is the complementary sequence to ATCCTA where A = adenine, C = cytosine, G = guanine and T = thymine.]

! Extension: The DNA polymerase[DNA polymerase: Any enzyme with the ability to synthesise new DNA strands, using a DNA template.] synthesises a complementary strand. The enzyme [Enzyme: A protein that functions as a catalyst of biochemical reactions.]reads the opposing strand sequence and extends the primers by adding nucleotides [Nucleotide: A molecule composed of a nitrogen base, a sugar and a phosphate group. Nucleotides are the building blocks of nucleic acids.]in the order in which they can pair. The whole process is repeated over and over。

The DNA polymerase, known as 'Taq polymerase', is named after the hot-spring bacterium Thermus aquaticus from which it was originally isolated. The enzyme can withstand the high temperatures needed for DNA-strand separation, and can be left in the reaction tube.

The cycle of heating and cooling is repeated over and over, stimulating the primers to bind to the original sequences and to newly synthesised sequences. The enzyme will again extend primer sequences. This cycling of temperatures results in copying and then copying of copies, and so on, leading to an exponential increase in the number of copies of specific sequences. Because the amount of DNA placed in the tube at the beginning is very small, almost all the DNA at the end of the reaction cycles is copied sequences.

The reaction products are separated by gel electrophoresis[Electrophoresis: A technique for separating the components of a mixture of molecules (proteins, DNA or RNA) by size as a result of an electric field within a support gel. Depending on the quantity produced and the size of the amplified fragment, the reaction products can be visualised directly by staining with ethidium bromide or a silver-staining protocol, or by means of radioisotopes and autoradiography.[Autoradiography: A technique where radioactively labelled molecules are visualised through exposure to X-ray film.