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

394 McAleer, Coffey, and Dunham
Fig. 1. A schematic representation of the vectorette method. Solid boxes represent genomic
DNA, and the hatched boxes represent YAC vector sequence. YAC DNA is digested with a
restriction enzyme, X. After ligation to annealed vectorette oligos, products are amplified
with a vectorette-specific primer (P2) and a primer specific for one or other of the YAC vector
arms (P1). Only fragments containing vector/insert junction are amplified. Confirmation of the
presence of the cloning site (CS) within the amplified fragment can be obtained by digestion
of the hybrid fragment with the enzyme that cuts at the cloning site, releasing a fragment
diagnostic of the vector arm (Table 2) together with one or more fragments corresponding to
the genomic DNA insert. CS = cloning site.
vector arms) in combination with a linker-specific primer. The linker-specific primer
corresponds to the sequence of the linker ligated to the 5′-end of each DNA strand and
has no complement on the other strand of the “bubble.” It is therefore unable to anneal
to template until the complementary sequence has been generated by priming off the
vector-specific sequence. Thus, only those fragments containing binding sites for the
vector-specific primer (i.e., DNA including and immediately adjacent to the cloning
site of the YAC vector) will be successfully amplified by the PCR. The amplification
products may then be used as DNA probes, for DNA sequencing, or may be cloned
into a suitable vector.
A recent adaptation of the vectorette method has been used to isolate possible
gene fragments from selected regions of the genome without prior knowledge of gene
sequence (7). This method is termed Island Rescue PCR (IRP), and relies on the fact
that nearly all housekeeping genes and over 40% of tissue-specific genes have a CpG
island in or near the 5′-end of the gene (8). Such CpG islands have a significantly
increased C + G content compared with the bulk of genomic DNA. These CpG