Therefore, our failure to identify wildtype sequence by direct sequence in TGK210Δ hearts suggested that a larger proportion of the transcript was mutant.
Molecular work-up based on polymerase chain amplification followed by direct sequence analysis of selected coding sequences of KIT (exons 9,11, and 13) and platelet-derived growth factor receptor α (PDGFRα; exons 12,14, and 18) yielded no evidence of mutation.
Analysis of PRNP sequences was performed by direct sequencing in a MacBAC sequencer (Pharmacia, USA).
Furthermore, the amplified products were isolated by electrophoresis on a 1% agarose gel and then purified using the QIAamp purification kit (Qiagen Inc ., and the nucleotide sequence was determined by direct sequencing using an ABI 310 automatic sequencer according to the manufacturer's instructions (Applied Biosystem, Foster City, CA, USA).
All samples with an abnormal curve were re-amplified and sequenced by direct sequencing using BigDye Terminator Cycle Sequencing Kit and ABI 310 automated sequencer (Applied Biosystems, Foster City, CA, USA).
For the samples presenting an aberrant peak on DHPLC analysis, the nucleotide sequence was determined by direct sequencing of PCR products (ABI 3100 DNA sequencer, Applied Biosystems).
PCR-amplified P510S sequence specificity was checked by direct sequencing of the amplified product by the automated DNA sequencer A.L.F.
Sequence identity was confirmed by direct sequencing of PCR products using an Applied Biosystems 3730 capillary DNA sequencer (Applied Biosystems, Foster City, CA, USA).
Finally, obtained sequences were verified by direct sequencing, using sequencing primers designed to cover target regions in both directions.
RT-QPCR primers were designed from the respective sequences and confirmed by direct sequencing as described.
The amplicons were screened for sequence variations by direct sequencing using the Big-Dye Terminator v3.1 sequencing kit (Applied Biosystems, Milan, Italy) and ABI 3130 Genetic Analyser (Applied Biosystems, Milan, Italy).
