PCR amplification techniques

Rosenthal, A. (1992) PCR amplification technique for chromosome walking. Trends Biotechnol. 10, 44-48. 

The most widely used amplification technique is the polymerase chain reaction. Hardly a research study in molecular biology involving DNA amplification has been performed without using PCR. Many modifications of PCR have been described. Before we review these modified techniques, it would be appropriate to briefly review the PCR methodology. 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction (PCR). The techniques for chemical synthesis of oligonucleotides have been highly developed. Very efficient and automated methodologies based on synthesis on a solid support are used widely in fields that depend on the availability of defined DNA sequences.152... 

PCR amplification has led to more sophisticated and accurate diagnostic techniques regarding diseases. This allows earlier detection of the disease compared to conventional methods, making earlier treatment possible. For example HIV (human immunodeficiency virus) may be detected by searching for the DNA sequence unique to this virus. Amplifying samples and searching for DNA associated with the bacteria responsible for the condition has identified infectious bacterial diseases. Lyme disease, certain stomach ulcers, and middle ear infections have been detected in this manner. 

If a mutation does not create or remove a restriction site it is usually possible to artificially create restriction sites during PCR amplification. The technique is referred to as amplification-created restriction site (ACRS) analysis and was first described independently by several groups in 1989 [6, 8,10]. It has since been applied to mutation analysis for many disorders. In the technique, one PCR primer directly... 

Troutt A. B., McHeyzer-Williams M. G., Pulendran B. and Nossal G. J. (1992) Ligation-anchored PCR a simple amplification technique with single-sided specificity. Proc. Natl. Acad. Sci. USA 89, 9823-9825. 

PCR is a very powerful technique, providing a sizable amount of DNA from a trace of a DNA sample. Hence it would be natural to expect that the trace amount of starting DNA can be quantitated sensitively from the amount of the finally-obtained PCR product. However, this type of quantitation based on the end-point detection is not reliable because of the saturation effects of PCR. This problem has been overcome by real-time PCR, which monitors PCR amplification in real time and enables accurate quantitation from the kinetics of the exponential phase. Real-time PCR thereby provides a highly sensitive and specific quantitation method for nucleic acids. 

We routinely clone chemokines using polymerase chain reaction (PCR). The small size of chemokine genes, the frequent addition of N-terminal or C-terminal tags, and the need to localize precisely the initiator methionine codon in bacteria expression systems are all most easily dealt with by amplifying the genes. In this section, it is assumed that the reader has a grasp of basic cloning techniques (18) and is familiar with simple PCR amplification (21). 

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