PCR technique

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. 

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

The PCR technique is very useful during all stages of the research and development of biotech crops. PCR analysis is used for gene discovery, event selection, screening, transformant identification, line selection and plant breeding. Quantitative real-time PCR is used to determine the number of transgene copies inserted in experimental... 

Current methods for DNA detection usually require enzymatic amplification of the target DNA sequence prior to analysis. For example, the PCR technique selectively increases the concentration of the target sequence relative to unrelated sequences. PCR methods, however, introduce ambiguities resulting from contamination by different DNA sequence. Therefore, a definitive method is required for the analysis of a single, original DNA sequence. To achieve this objective, the sensitivity and speed of the chemiluminescent enhancement techniques described in this chapter must be improved. 

To minimize problems with the detection and analysis of a gene that exists as a single copy on an autosomal chromosome, technology of extreme sensitivity needs to be employed. Although the standard Southern analysis combines reasonable sensitivity with greater specificity, it is labor-intensive, requiring the use of radioisotopes such as 32P, and a few days are required to complete an analysis. Several pitfalls of the Southern procedure can be eliminated by substituting the PCR technique (M4). 

There are two different approaches to performing the in situ reverse transcription (RT)-PCR technique (22-25). 

Another popular technique for endophyte identification is RFLP (restriction fragment length polymorphism).In this technique, nuclear or mitochondrial DNA is extracted from the endophyte and digested with restriction endonucleases. The digestion products are separated by gel electrophoresis, and a unique pattern emerges for the fungus, similar to the patterns seen in the RAPD-PCR technique. 

Use hot-start tubes and assemble the bottom and top part of the reaction for second-strand synthesis and amplification of the DNA template by error-prone PCR. Hot-start PCR is the PCR technique of assembling the reaction mixture at a temperature that is greater than the annealing temperature. This procedure increases precision, yield, and specificity. The pre-adhered wax bead assures synchronous reaction start-up and eliminates the need for using mineral oil. 

The development of the polymerase chain reaction (PCR) technique made it possible to amplify isolated opioid receptor cDNA and to synthesize small amounts of the receptor protein necessary to identify their amino acid... 

Applications of the PCR technique include 1) efficient comparison of a normal cloned gene with an uncloned mutant form of the gene, 2) detection of low-abundance nucleic acid sequences, 3) forensic analysis of DNA samples, and 4) prenatal diagnosis and carrier detection, for example, of cystic fibrosis. 

The PCR technique provides a way of "amplifying" a small number of DNA molecules, i.e., to produce many copies. This is often done by cloning but PCR offers a quick and easy way to obtain millions of copies of a desired relatively short segment of DNA. Standard PCR can be used for up to about 5000-nucleotide pieces. More recently modified procedures have allowed 35-kb segments to be amplified.600... 

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