John M. S. Bartlett.pdf - Bio-Nica.info

The Vectorette Method 393
57
DNA Rescue by the Vectorette Method
Marcia A. McAleer, Alison J. Coffey, and Ian Dunham
1. Introduction
A major advance in physical mapping of the human genome was the development of
yeast artificial chromosome (YAC) vectors (1). This has enabled the cloning of pieces
of DNA several hundred kilobases in length (2). The availability of such large cloned
genomic DNA fragments means that by ordering a series of overlapping YAC clones,
a contiguous stretch of DNA, several megabases in length, can be isolated around a
genomic region of interest (e.g., the region of a chromosome linked to a particular
disease gene). The successful isolation of terminal sequences of a given YAC can be
very useful in assembling an ordered “contig” of YAC clones. Such terminal clones may
be used directly as hybridization probes or sequenced and used to generate sequence
tagged sites (STSs) to identify overlaps between, and isolate other, members of the
contig. Several methods have been used to this end, including PCR with vector-specific
primers in combination with primers designed either for repetitive elements, such as Alu
sequences (3), or in combination with random nonspecific primers (4). However, these
techniques rely on a suitable repetitive element or random primer sequence occurring
close enough to the end of the YAC so as to be amplified by PCR. Furthermore, probes
isolated in this manner may well contain highly repetitive sequences that, if unsuccessfully
blocked, will increase nonspecific signal in any subsequent hybridization
procedures (5).
The vectorette method was originally described by Riley et al. (6). YAC DNA is
digested with a restriction enzyme, and the resulting fragments are ligated to a linker
molecule to create a vectorette “library,” that is, a complex mixture of restriction
fragments with linker ligated to each end. Within this library are fragments that contain
the YAC vector/genomic DNA junction, which includes the terminal sequences of
the YAC (Fig. 1).
The linker molecule consists of two long (>50 nucleotides) preannealed oligonucleotides
incorporating a suitable 5′-overhang corresponding to the restriction enzyme
used in the initial YAC digest. Blunt-ended linkers may also be used. Although the
oligonucleotides comprising the linker are complementary at the 5′- and 3′-ends, there
is a region of noncomplementarity in the middle where the two strands are unable to
pair and a vectorette “bubble” is formed. The PCR is then performed on this mixture
using one of two vector-specific primers (designed either for the centric or acentric
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
393