The overall aim is to develop an extend our recent breakthrough discovery of a thermostable DNA polymerase formulation that can amplify up to 23 kb so far. The current template is lambda phage DNA. Concomitant with the newly achievable long lengths of PCR product, the fidelity is also as high or higher than any PCR amplification system so far described. Many areas of molecular biology, gene analysis, and gene mapping will benefit from this system, especially if it can be extended to at least 30-40 kb and if it can be further developed to make it applicable to genomic DNA of bacterial or human complexity. Improvements to the amplification system will be sought by experimenting with critical parameters while amplifying target sizes near the current edge of reliability (now 15-18 kb). As improvements are made, the test bed DNA spans will be longer. Experiments to extend the maximum length of achievable PCR product from lambda DNA template include; 1) Increase the length of primers to allow maximum temperature and maximum primer selectivity during the annealling/extension step of the PCR. 2) Increase the thermostability of the system, now surprisingly limited to only 20 seconds at 94% C during the denaturation phase of each PCR cycle. 3) Scan reaction buffer condiitons and cycling protocols. 4) Mutagenize one of the enzyme components of the mixture to test the theory of how it works, and to possible improve how it works. After simulated genomic DNA amplifications with reliable lambda DNA template mixed with human DNA, experiments to apply the system to genomic DNA analysis will utilize primers of 5, 10, 15, 20, and 25 kb separation on known sequenced regions of human DNA. A form of long primer to be tested will be STS megaprimers, since this syst em has also been shown to utilize such primers more effectively than other known thermostable DNA polymerases. Genomic maps defined by STSs will then benefit easily from this advance.
