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

398 McAleer, Coffey, and Dunham
Table 2
Positions of Primer Sequences Described in Table 1 with Respect
to the EcoRI Sequence in the Cloning Site of pYAC4
Centric arm
Acentric arm
1089→EcoRI 287 bp 1091→EcoRI 172 bp
Sup4-2→EcoRI 40 bp Sup4-3→EcoRI 29 bp
pYACL→EcoRI 17 bp pYACR→EcoRI 20 bp
10. A second PCR may be performed to reduce the amount of vector DNA contained in the
amplified product. A nested vector-specific primer that anneals closer to the cloning site
(Tables 1 and 2) is used in combination with the vectorette-specific oligo. Either use
1 µL of the primary PCR or toothpick the fragment found to cut with EcoRI in step 9
(not the restriction digestion product) directly from the agarose gel into a PCR containing:
for “left” arm products: Sup4-2 + 224 or pYACL + 224, and for “right” arm products:
Sup4-3 + 224 or pYACR + 224. The same cycling conditions as those described in step 8
are used, but the annealing temperature is reduced to 59°C and only 20 cycles are
performed. Ten microliters may be visualized on a 2.5% agarose minigel.
11. PCR products may now either be sequenced directly, radiolabeled and used as a hybridization
probe (see Note 5), or subcloned using a suitable cloning system, such as pCR-Script
SK (Stratagene) or TA-cloning system (Invitrogen, Leek, The Netherlands).
4. Notes
1. Use approx 1 µg of solution DNA for each restriction enzyme digest. Reactions should be
performed in the buffers recommended by the manufacturers for 4 h at the specified
temperature. Before ligation (Subheading 3., step 5), enzymes should be heat-inactivated
(65°C for 15 min is usually sufficient), extracted with phenolchloroform (equal volume of
ratio 11), ethanol-precipitated by standard methods (2 vol 95% ethanol with one-tenth vol
3 M sodium acetate, pH 5.6) and resuspended to a concentration of 250 ng/µL. Ligations
can be performed in a volume of 10 µL with 1 µL preannealed vectorette oligos.
2. It is important to check that there are no recognition sites for a given restriction endonuclease
between the sequences corresponding to the vector-specific primers and the
cloning site. If such a site were present, it would be cleaved in the initial digest and a
vector-only fragment would be amplified. Suitable enzymes for pYAC4 are RsaI, PvuII,
and StuI.
3. The addition of Perfect Match to the PCR reduces the number of nonspecific bands generated
(compare Fig. 2A with B), although some laboratories have found little difference
on its omission.
4. IRP is a variant of Alu-vectorette PCR that can be used to generate probes from YACs as an
alternative to Alu-PCR. For IRP, the universal vectorette primer 224 is used together with
primer sequences that recognize a human Alu repeat. For example: 5′-GGATTACAGGC-
GTGAGCCAC-3′ and 5′-GATCGCGCCACTGCAC TCC-3′ (both sequences taken from
ref. 7). The thermocycling conditions described in Subheading 3., step 8 may also be
used for these two sets of primers.
5. Probes generated by this method may contain highly repetitive sequences. Therefore, it is
advisable to pre-reassociate the labeled probe with total human genomic DNA prior to any
hybridization procedure. Make probe up to 250 µL with H 2 O. Add 125 µL of 10 mg/mL
sonicated total human DNA (Sigma) and boil for 5 min. Snap-chill on ice for 5 min, and
then add probe to hybridization as normal.