Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Improvement of Ligation-Mediated PCR for DNA Sequence Determination

Kenneth S. Graham, Arian F.A. Smit, and Arthur D. Riggs
Division of Biology, Beckman Research Institute of the City of Hope, Duarte CA 91010

We have been investigating the use of ligation-mediated PCR (LMPCR) as an approach to sequence determination of DNA that is difficult or impossible to clone, since LMPCR enables direct sequence determination of total genomic mammalian DNA. The basic method consists of 1) treatment of the genomic DNA with sequence specific cleavage agents, 2) primer extension with a gene specific oligonucleotide 3) ligation of an oligonucleotide to the blunt ends, 4) exponential PCR using a gene specific primer and a linker specific primer, and 5) sequencing analysis of the PCR products. All areas of the technique have been examined and improved, resulting in a 30% increase in overall speed, improved specificity and use of non-radiolabeled detection. In the area of sequence specific cleavage agents, a major improvement was achieved by incorporating an A specific cleavage protocol. Both primer extension and PCR were improved through the use of thermal stable polymerases such as Vent (exo-) for first primer extension and then Vent for PCR. The greater thermal stability of these enzymes allowed the use of higher Tm primers which give increased specificity and lower background. Since these enzymes have greater processivity than the previously used Sequenase and Taq, longer reads have been achieved and the sensitivity to GC rich sequences has been reduced. Unambiguous determination of previously unknown sequences was accomplished. Moreover, the increased specificity and lower background also allowed the use dye conjugated primers for nonradioactive product visualization by use of an ABI automated sequencer.

Investigation was begun of a potentially major improvement using ligation of linker to the 3' end of primer extended molecules instead of, as in standard LMPCR, to the 5' end of molecules rendered blunt-ended by primer extension. Initial experiments were done using T4 ligase and were promising, but efficiencies were low, so we have been investigating a novel chemically mediated ligation procedure which should increase the efficiency of 3' ligation.

Sequence determination by LMPCR is essentially a primer-walking method, so it ultimately depends on the specificity of oligonucleotide primers. We observed, initially at the DHFR locus, that some primers gave smears even though they were expected to be for single-copy target sequences. This work led to the discovery of a new, abundant family of mammalian apparent LTR retroposons (MaLRs) [1]. This analysis of repetitive sequences has now been expanded to cover both SINES and LINES. Consensus sequences for several repetitive element families have been derived and have been contributed to the repetitive sequence database being compiled by J. Jurka.

This work was funded by the DOE Genome Program (ER6-1137, A D. Riggs P.I.)

[1] Smit, A.F.A. (1993) Identification of a new, abundant superfamily of mammalian LTR-transposons, Nucleic Acids Res. 21: 1863 1872.