Genomic DNA was prepared from peripheral blood by
phenol-chloroform extraction or using the QIamp blood kit (Qiagen,
Santa Clarita, CA) according to manufacturer’s instructions.
Polymerase chain reaction (PCR) amplification was obtained from genomic
DNA using previously published sets of primers.
14 After
PCR amplification, fragments were analyzed on a 8% polyacrylamide gel
and visualized by ethidium bromide staining. To sequence the amplimers
individually, these were separated on a 1.6% low-melting-point
agarose, and the bands were excised and treated withβ
-agarase and subsequently phenol-chloroform purified. The
purified products were used for direct manual sequencing using a cycle
sequencing kit with
33P-labeled dideoxy
nucleotides (Thermo Sequenase; Amersham, Life Sciences, Arlington, IL),
according to the manufacturer’s instructions. To further confirm the
mutations, the gel purified amplimers were cloned into the pCR 2.1-TOPO
vector, using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA)
according to the manufacturer’s recommendations. For each of the two
probands, 10 different amplimer subcloned fragments were sequenced at
the Johns Hopkins University core facility, using the ABI 100, version
3.2 automated sequencer (ABI prism; Applied Biosystems, Foster City,
CA).