Gene synthesis, polymerase chain reaction

Synthesis of Genes by Polymerase Chain Reaction. Another strategy that takes advantage of specific oligonucleotide primers is the in vitro amplification of specific DNA fragments from very small amounts of an impure sample. Since the first demonstration of the technique by Mullis and Faloona (57) it has become one of the most popular procedures in molecular biology and virtually has transformed our approach to isolation of genes [for review see (58-60). 

The use of molecular probes to track specific microbes in the environment, specifically those not easily cultured, has been recently reviewed (95, 97, 98), The sensitivity of these probes may be further enhanced by using amplification strategies (e.g., polymerase chain reaction or PCR), to amplify segments of DNA from samples obtained from production systems (95, 99), However, gene probes for geosmin or MIB synthesis are not currently available. 

Downstream application [PCR (polymerase chain reaction), cloning, labeling, blotting, RT (reverse transcriptase)-PCR, cDNA synthesis, RNAse protection assays, gene therapy, etc.]... 

The solid-phase synthesis of DNA oligomers has reached a high level of maturity and is extensively used for the synthesis of primers for polymerase chain reaction or the design of new genes (13). In contrast to the cellular process, the synthetic route forms the polymer in 3 -to-5 direction. The oligomerization is carried out on CPG using a base labile linker. Typically, the nucleobases are introduced as acyl-protected phosphoramidites,... 

Figure 4. Diagramatic representation of the polymerase chain reaction (PCR) used to amplify particular DNA sequences. At the top are DNA molecules present in a mixture of bulk DNA with the heavy lines representing the gene of interest. In step A the DNA is melted at elevated temperature after which oligonucleotides with a sequence complementary to the DNA of interest (B) are added. The oligonucleotides act to prime the synthesis of new DNA complementary to the DNA of interest by a heat stable DNA polymerase (C). After an adequate period of time the DNA is remelted at elevated temperature and the cycle is repeated (D).

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