DNA extraction
Genomic DNA was extracted from peripheral blood of the proband using DNA
extraction kits and standard protocols (TSP201-50, Tsingke, China).
Sanger sequencing
For analysis of UBE2A splice site mutation, the intron-based exon
specific primers were designed with Primer 5 (primer-F:
TGGGCCCAGTTTCTTAAGGA; primer-R: AATCAGGAGGCTCCCAGACT). The primers were
used for amplify UBE2A exons. PCR was amplified with FastStart Taq DNA
Polymerase, dNTPack (Roche) and
purified with DNA Gel Extraction Kit
( GE0101-50, Tsingke, China)
following standard protocols. BigDye Terminator v3.1 Cycle Sequencing
Kit was used for sequencing reactions prior to sequencing on a
48-capillary 3730 DNA Analyzer (Applied Biosystems).