DOP-vector PCR: a method for rapid isolation and sequencing of insert termini
from PAC clones
DOP-vector PCR: a method for rapid isolation and sequencing of insert termini from PAC clones
Chenyan
Wu
,
Shigui
Zhu
,
Stacey
Simpson
and
Pieter J.
de Jong*
Department of Human Genetics, Roswell Park Cancer Institute,
Buffalo
, NY 14263,
USA
Received April 1, 1996;
Accepted May 21, 1996
To develop a physical map, one often uses a large-insert library to establish a contiguous set of overlapping genomic clones
(`contig'). One of the most efficient ways to prepare contigs in limited
chromosomal regions is chromosome-walking. For this approach, it is important to retrieve DNA sequences from
the termini of the insert fragment to screen for overlapping clones. Many
methods for the isolation of DNA insert termini have been developed: subcloning
(
1
), inverse PCR (
2
), Alu-vector PCR (
3
), bubble-vector PCR (
4
), adapter-vector PCR (
5
), junction trapping (
6
), capture-PCR (
7
), TAIL-PCR (
8
) and direct sequencing of the end fragments (
9
). Many of these methods work well but are difficult to scale up to large
numbers of end-rescue reactions. Our goal was to improve the TAIL-PCR method, such that nearly every insert end from P1-derived artificial chromosome (PAC) clones (
11
) can be amplified directly from bacterial suspension, thus avoiding the cost of
plasmid DNA extraction.
Here we present an efficient method, termed DOP-vector PCR, to amplify unknown terminal sequences from PAC clones. This
technique should also be applicable to other systems, e.g. BAC or YAC clones.
The basic concept is to employ a partially-degenerate oligonucleotide primer (DOP) in combination with a vector
primer (Fig.
1
). The partially degenerate primer 6-MW (CCGACTCGAGNNNNNNATGTGG) has been described previously (
12
). It has six bases specified at the 3'-end, six completely degenerate bases in the middle, and an
arbitrary 10 nt sequence at the 5'-end. A single cycle of DNA synthesis is carried out at a low stringency annealing temperature
in the presence of the 6-MW primer. Primer extension is catalyzed by the modified T7 DNA polymerase
(Sequenase version 2.0, USB) as has been reported previously (
13
). Under these low stringency conditions, priming and subsequent DNA elongation
probably occurs from numerous discrete sites in the PAC DNA (and the
Escherichia coli
DNA), which match for at least 5 or 6 nt with the 3' terminal nucleotides of the DOP primer. For our application, it is
essential to permit random priming while preventing random amplification. Therefore, only a single cycle of low stringency DNA synthesis is allowed. To
selectively amplify the vector/insert junction sequences, high-stringency PCR conditions are imposed using a vector primer in combination
with the DOP primer and using
Taq
polymerase for DNA synthesis. During the first PCR cycle, a subset of the
nascent DNA strands (from the Sequenase synthesis) are used as templates for
DNA synthesis primed by the vector primer. The products include, for the first
time, the complementary sequence for the DOP primer, which can serve as a high stringency priming site for the 6-MW primer in subsequent PCR cycles. The specificity for junction fragments
can be further enhanced by a second PCR employing a nested vector primer in
combination with the DOP primer (Fig.
1
D). Please note that the anticipated junction PCR fragments generated from the
first PCR reaction will be truncated in the second PCR reaction with nested
primers. This will result in a shifting of the PCR mobility of 73 bp for the T7
junction and 78 bp for the SP6 junction.
REFERENCES
1 Traver,C.N., Klapholz,S., Hyman,R.W. and Davis,R.W. (1989) Proc. Natl. Acad. Sci. USA,86, 5898-5902.MEDLINE Abstract
2 Ochman,H., Gerler,A.S. and Hartl,D.L. (1988) Genetics,120, 621-623.MEDLINE Abstract
3 Nelson,D.L., Ledbetter,S.A., Corbo,L., Victoria,M.F., Ramirez-Solis,R., Webster,T.D., Ledbetter,D.H. and Caskey,C.T. (1989) Proc. Natl. Acad. Sci. USA, 86, 7485-7489.MEDLINE Abstract
