Simultaneous Bi-Directional Cycle Sequencing.

Stephen C. Roemer, Kurt A. Brumbaugh, Vince Boveia, Mary Jensen, and John Gardner¹
LI-COR, inc., Biotechnology Division, 4647 Superior St., Lincoln, NE 68504 and ¹CEPRAP, University of California, Davis, CA

Requires Less DNA Template

For smaller inserts and PCR products (<1.5 kb), less DNA template is needed when cycle sequencing bi-directionally as compared to traditional single-primer cycle sequencing. This is due to the quasi-PCR kinetics established during a bi-directional cycle sequencing reaction in which some primer extensions will reach to the opposite priming site thus serving to amplify the original amount of DNA template.

A 360 bp PCR product was cycle sequenced bi-directionally using Thermo Sequenase, two custom primers specific to the PCR product, and different amounts of template (1 - 40 fmole). Readable sequence ladder was generated even at 1 fmole of DNA template, which is about 50-fold less than the amount required for a single-primer cycle sequencing reaction. Samples are loaded ATGC and data from the 800 channel are shown.

Provides the Longest Single Sequence Coverage

More than 2000 bases (no overlap with inserts > 2.4kb) can be obtained simultaneously from a single bi-directional cycle sequencing reaction using a 66 cm, 4% Long Ranger gel on the LI-COR IR² Automated DNA Sequencing System.

SBS reactions were performed using Thermo Sequenase, 400 fmole of an 11.5 kb plasmid (pBluescript II SK(+) with 8.5 kb insert from rat genomic DNA), and 1.5 pmole of both IRD700-T3 Promoter and IRD800-T7 Promoter primers. Sections of the T3 reaction are shown in standard chromatogram format (SCF). The autosequencer called 1,011 bases for the T3 primer and 999 bases for the T7 primer.

Confirms Mutations

To achieve high accuracy in sequencing-based heterozygote detection, it is necessary to sequence the forward and reverse DNA strands. Twelve fmole of a 260 bp PCR product was cycle sequenced bi-directionally using SequiTherm Excel II DNA polymerase and 1.7 pmole of each strand-specific primer. The AC heterozygote at base position 109 of the forward strand is easily confirmed using the sequence of the complementary strand (TG at base position 102).

Resolves Ambiguities

Resolving band compressions on DNA sequencing gels hinders the high-speed and accurate determination of DNA sequences. Bi-directional cycle sequencing generates confirmatory sequence in a single reaction so that compressions in one strand are quickly resolved using sequence from the complementary strand.

An example of a sequence motif (5' - CGCAG - 3') causing band compression (3) is shown in the forward strand of a 3.4kb plasmid. The reverse sequence produced during the SBS reaction clearly resolves the compression.