L D Ohler | PubMed Health

PubMed1993

Use of a sensitive fluorescent intercalating dye to detect PCR products of low copy number and high molecular weight.

The ability to routinely and specifically amplify and detect PCR products ranging in size from < 1 to > 10 kb, regardless of target template sequence or structure, would facilitate several tasks in human genome research. Generation of a wide size range of PCR products would potentially expedite isolation of uncloned DNA (gaps) represented in physical maps and provide a means for maintaining order and orientation of closely linked loci during analysis. Long-range PCR offers an alternative to isolation of genomic or cDNA clones from tissues or species where appropriate libraries are unavailable. This methodology also provides a powerful strategy to pursue directed transposon-based mapping and sequencing of templates of 100 kb or greater. Further development of basic PCR technology has been necessary in order to make it feasible to use large DNA targets as primary templates for genome analysis. Application of these extended PCR capacities would potentially save time, materials, and cost.

Bacteriophage lambda↗

PubMed1992

Optimization of long-distance PCR using a transposon-based model system.

The ability to amplify routinely long PCR products (5-25 kb) with high specificity and fidelity, regardless of target template sequence or structure, would provide significant benefits to genome mapping and sequencing endeavors. Although occasional reports have described the generation of long PCR products, such results have been difficult to replicate and have frequently utilized probe hybridization to identify the specific product from nonspecific amplified DNA. Production of specific PCR products has generally been limited to target templates of less than 3 kb. To extend the effective range of standard PCR amplification, it may be necessary to utilize alternative reaction conditions and/or components, such as novel thermostable DNA polymerases or accessory proteins. We describe the use of a model system to evaluate systematically methodological changes that might enable efficient long-range PCR. Specifically, the transposon Tn5supF has been used to introduce randomly identical, known primer binding sites within separate isolates of phage clones carrying identical inserts. Transposon-based PCR allows us to study amplification of DNA fragments that vary in size and sequence using only a single set of primers. In the present studies, we describe conditions that enable PCR amplification of specific DNA templates ranging in size up to 9 kb. Some of the key features of our methodology include the use of recombinant Thermus thermophilus (rTth) DNA polymerase, the addition of gelatin to the reaction mixture, the use of wax-mediated "hot starts" and, lastly, the use of auto-segment extension thermocycling. These results also provide insights into additional approaches that might further enhance our ability to perform long-distance PCR.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗