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

The Vectorette Method 395
islands can be detected in native human genomic DNA, by rare-cutting restriction
endonucleases that recognize unmethylated CpG-containing sequences. The principles
of the vectorette method described above are used except the YAC DNA in this instance
is digested with restriction endonucleases that specifically recognize CpG-containing
sequences, for example, SacII, EagI. Therefore, YAC DNA will be cut at CpG-rich sites,
which may be associated with a gene. The mixture is then ligated to the preannealed
vectorette oligos, and PCR in this instance is driven by an Alu-specific primer together
with the vectorette oligo described above. Northern blot analysis may then be used
to test that amplified sequences are associated with expressed mRNAs. There are
two main drawbacks to this method. First, because DNA in yeast is not differentially
methylated, all CpG-containing restriction sites will be cut whether or not they are
associated with an unmethylated island in native genomic DNA. Therefore, a portion
of the amplified fragments may not be associated with an expressed mRNA. Second,
as with all Alu-PCR based methods, there is a requirement for an Alu sequence close
enough to the restriction site to allow amplification by the Taq polymerase. However,
in terms of transcript mapping, where the previously described methods, for example,
direct selection/cDNA enrichment (9), exon trapping (10), probing cDNA libraries
directly with radiolabeled YAC DNA (11), all have limitations, IRP may prove to
be a rapid and useful technique for the identification of transcriptional units within
complex sources of DNA.
Although the vectorette method was originally developed for rescuing the vectorinsert
junctions of YACs, it may be used to isolate sequences adjacent to any known
sequence, for example, the identification of intron/exon boundaries in a specified
gene (12). This chapter describes in detail the application of the vectorette method to
isolating terminal sequences from YACs.
2. Materials
All solutions should be made to the standard required for molecular biology, that is,
using sterile distilled water and molecular-biology-grade reagents.
1. T4 DNA ligase, 1 U/µL and 5X T4 DNA ligase buffer (0.25 M Tris-HCl, pH 7.6, 50 mM
MgCl 2 , 5 mM ATP, 5 mM DTT, 25% [w/v] polyethylene glycol-8000; Gibco BRL, Paisley,
Scotland).
2. The sequences of the oligonucleotides used in this chapter are given in Table 1 and are
taken from ref. (13). The vector-specific primers are designed against pYAC4 (these can
be replaced with appropriate vector primers or Alu-specific primers if performing IRP).
The vectorette oligonucleotides described are suitable for blunt-ended ligations. If desired,
a suitable overhang at the 5′-end of the “top” strand oligonucleotide may be incorporated
to facilitate “sticky ended” ligations.
Oligonucleotides were synthesized by phosphoramidite chemistry on an Applied
Biosystems 392 DNA/RNA synthesizer. After deprotection (7 h at 55°C), oligonucleotides
are dried in a centrifugal evaporator (alternatively, the standard ethanol precipitation
procedure may be used). Oligonucleotides used in PCR are resuspended in H 2 O to a
concentration of 20 µM. Vectorette oligonucleotides are purified by high-performance
liquid chromatography (12% polyacrylamide gel electrophoresis may also be used). Before
use, equimolar quantities of the “top” and “bottom” oligonucleotides are preannealed in
25 mM NaCl by heating at 65°C for 5 min and left to cool to room temperature. A working
concentration of 1 µM is used in ligations. All oligonucleotides are stored at –20°C.