Genomic DNA was prepared from peripheral blood as
described.
8 The 15
TULP1 exons were amplified
in polymerase chain reaction (PCR) by using primers anchored in the
corresponding flanking intron sequences (sequences and PCR conditions
were kindly provided by Alan Wright).
Amplification in a thermal cycler (Perkin–Elmer Applied Biosystems,
Foster City, CA), was performed in a total volume of 50 μl. Each
reaction contained 100 ng genomic DNA, 20 picomoles of each primer, 200μ
M dNTPs, and 1.25 U Taq polymerase (Promega, Madison,
WI) in a buffer containing 1.0 to 1.5 mM MgCl2 with or
without 10% dimethyl sulfoxide. Reactions were generally subjected to
35 cycles of 94° for 40 seconds, X° for 30 to 40 seconds (where
X° is the annealing temperature between 52° and 60°), and,
optionally, 72° for 30 seconds. A final extension step was performed
at 72° for 5 minutes.
Single-strand conformation polymorphism (SSCP) analyses were performed
as described previously.
9 Each PCR-amplified fragment was
assayed under three different conditions, combining acrylamide and
glycerol concentrations and running temperatures. The DNA sequence was
obtained for each PCR sample showing an aberrant SSCP pattern.
Sequencing was performed directly on PCR products using a dye
terminator cycle sequencing premix kit (Thermosequenase; Amersham
Pharmacia Biotech, Uppsala, Sweden). In addition, PCR products of exons
5 and 10 of the index patient were cloned using a ligation kit
(Sureclone; Amersham Pharmacia Biotech), and clones were sequenced as
described.
The deletion IVS4–2delAGA was confirmed by MspI
digestion after PCR amplification, by using a forward primer with a
mismatch (italics): 5′ TTATGAAGAGTTTCTACCTCC 3′, which
generated an MspI restriction site in the mutant allele.