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.

ONE-STEP PCR SEQUENCING

Ken Porter, David Briley, and Barbara Ramsay Shaw*
Department of Chemistry, Duke University, Durham, NC 27708

A method is described to simultaneously amplify and sequence single- or double-stranded DNA. The method, which we call One-Step PCR Sequencing, is unique in that it employs a new class of alphaP-borane 2'-deoxynucleoside 5'-triphosphates (dNT(b)Ps) first synthesized in our laboratory. These boronated triphosphates exhibit useful properties: (a) they are heat stable, (b) they can be incorporated, base-specifically, into DNA during the polymerase chain reaction, and (c) once incorporated, the boranophosphate nucleotides block the action of exonuclease III. For One-Step Sequencing, a small percentage of boranophosphates are incorporated into DNA during PCR, and the positions of the stably-incorporated boronated dNMPs can be revealed by a simple exonuclease digestion, thereby defining the sequence of the PCR product. The One-Step method should eliminate two costly and time-consuming steps associated with current PCR sequencing techniques: (a) DNA purification following amplification and (b) single-sided primer extension with dideoxynucleotide chain terminators. As a consequence, the One-Step method should both decrease the time required to sequence PCR products, and render PCR sequencing completely automatable. Further, since the boranophosphate sequence delimiters are incorporated into the DNA during PCR, the method is readily amenable to bidirectional sequencing.

We have performed several preliminary experiments which are described below.

A. PCR is not inhibited by the presence of a small percentage of dNT(b)Ps. In fact, the dNT(b)Ps are incorporated extremely well into the PCR products and render the PCR products resistant to exonuclease III.

B. Primers can be extended in the presence of 100% of one boronated dNTP (A, T, G, or C) plus the three remaining normal dNTPs. These extension products are resistant base-specifically to exonuclease III under conditions in which all-normal extension products are degraded completely. Also, the presence of boronated nucleotides in these oligomers does not affect their electrophoretic mobility in 16% polyacrylamide gels.

C. One-Step PCR Sequencing was demonstrated with an end-labeled primer. The 509 bp region between phage T7 positions 34534 and 35042 was amplified by PCR using one labeled and one unlabeled PCR primer in the presence of a small percentage of each dNT(b)P. The PCR products were digested with exonuclease m and separated by PAGE. Approximately 350 bases could be read from each strand.

D. Bidirectional One-Step PCR Sequencing was demonstrated with a biotinylated primer. The 629 bp region between T7 positions 21786 and 22414 was amplified by PCR using one biotinylated and one unmodified PCR primer in the presence of alpha-33P-dATP and each dNT(b)P. The products were incubated with streptavidin-linked magnetic beads, immobilized with a magnet, and digested with exonuclease m. Fragments from both strands were isolated and separated by PAGE. Sequencing data over 200-300 bases with boranophosphates compared favorably with dideoxy cycle sequencing.

Our method is rapidly approaching the quality of data which can be produced by cycle sequencing, yet requires much less DNA than the standard cycle-sequencing reaction. More DNA is required for cycle sequencing because the dideoxy-NTP chain truncators limit product formation to a linear accumulation. In contrast, One-Step PCR sequencing requires minimal starting template DNA because the boranophosphate sequence delimiters permit exponential PCR amplification. The key advantages of boranophosphates as sequence delimiters in PCR are that they (1) delineate the DNA sequence and (2) do not obstruct exponential amplification.