Introduction to the Workshop
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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.
Sequence-Directed DNA Manipulation for Enhanced Mapping and Sequencing
Charles R. Cantor, Natalia Broude, Takeshi Sano, Kazuhiro Tsukamoto, Marek Przetakiewicz, A. Burak. Dalan, Abha Chandra, Rhonda Harrison, Nikolay Bukanov, Ronald Yaar, Demetri Moustakas, Przemekyslaw Szafranski, and Cassandra L. Smith.
Center for Advanced Biotechnology and Department of Pharmacology, Boston University, College of Engineering, Boston MA 02215. Corresponding author.
Most traditional DNA analysis is done based on fractionation of DNA by length. We have, instead, begun to explore the use of DNA sequences as capture and detection methods to expedite a number of procedures in genome analysis. In enzyme-enhanced sequencing by hybridization (SBH), a partially duplex DNA probe is used to capture the five complementary bases at the end of a single-stranded DNA target. DNA ligase can be used to covalently seal the target to the probe and ensure faithful detection of the target sequence. Subsequent DNA polymerase I extension of the probe, now serving as a primer, along the target, now serving as a template, allows additional bases of DNA sequence to be read and also further ensures the fidelity of the original detection. Pilot studies show that this procedure can, indeed, reveal the sequence of a DNA target. By incorporating length information about the target, many of the branch point ambiguities that occur in ordinary SBH can be eliminated.
While conceived as a direct DNA sequencing procedure, the SBH format we use may be even more useful as a device for the rapid preparation of DNA samples for fast serial methods like capillary arrays or mass spectrometry. For example, an array of only 1024 probes could capture and then generate sequence ladders from any arbitrary DNA sequence. Some of the ways in which this sort of capture device might be used in DNA sequencing will be illustrated. We have also begun to explore the use of sequence-specific DNA capture as a method for looking at specific DNA sequence differences. This could eventually lead to faster methods for genetic mapping. For effective sequence-specific capture it is vital that non-specific binding of target DNA to the substrate used for the probe array be minimized. We have been exploring a number of different surfaces and modes of attachment to try to achieve the very low backgrounds needed to handle large numbers of samples simultaneously. Some of the probe designs we are using would allow direct production of probe arrays by replication of a master array and transfer to a new surface.
Human-specific PCR is widely used in DNA mapping. The most common version of this technique is inter-Alu PCR. Two major limitations in this approach are the relatively small fraction of a human target sample that is amplified and the poor results generally obtained from regions of the genome in which Alu sequences are relatively sparse. Many of these problems would be eliminated if a relatively efficient single-sided Alu PCR procedure were used, instead of inter-Alu PCR. We have developed such a procedure based on asymmetric PCR between an Alu primer and a ligated splint. The new procedure appears to give much better coverage when human YAC DNA is amplified. We have devised and tested several other new amplification methods including an efficient procedure for relatively uniform whole genome amplification and a non-enzymatic spatially-resolved DNA amplification method that might assist the analysis of sample arrays or chromosome spreads.