Online Appendix for the following JACC article
TITLE: Screening for Copy Number Variation in Genes Associated With the Long QT
Syndrome: Clinical Relevance
AUTHORS: Julien Barc, PHD, François Briec, MD, Sébastien Schmitt, Florence Kyndt,
PharmD, PHD, Martine Le Cunff, BS, Estelle Baron, BS, Claude Vieyres, MD, Frédéric
Sacher, MD, Richard Redon, PHD, Cédric Le Caignec, MD, PHD, Hervé Le Marec, MD,
PHD, Vincent Probst, MD, PHD, Jean-Jacques Schott, PHD
APPENDIX
Conditions and primer sequences for Quantitative Multiplex PCR of Short Fluorescent
Fragment (QMPSF)
Reactions were performed in a volume of 25 μl containing 0.2 mM each deoxynucleoside
triphosphate, 5 mM MgCl2, 7.5% DMSO, 100 ng of DNA, 0.2 mM PCR primers targeting
each tested exon, 0.1 mM primers targeting exon 14 of MLH1 gene, and 1.5 Units of
Platinum Taq DNA Polymerase with its buffer (Invitrogen). Every primer carried a 10nucleotide sequence extension at its 5′ extremity, each forward one carrying a [6-FAM]
group. Primers sequences are as follows:
KCNH2-ex5-F: [6-FAM]-CGTTAGATAGCGCACCATTAGCAAGATTCC,
KCNH2-ex5-R: GATAGGGTTATGTGGGTTCGCTCCTTTATC,
KCNH2-ex15-F: [6-FAM]-CGTTAGATAGGCAGGTTTCCCAGTTCATGG,
KCNH2-ex15-R: GATAGGGTTAGCTGTGCTTTCGAGTTCCTCTC,
KCNQ1-ex7-F: [6-FAM]-CGTTAGATAGCTGCAGGTCACAGTCACCAC,
KCNQ1-ex7-R: GATAGGGTTAGCAAAGAAGGAGATGGCAAA,
KCNQ1-ex8-F: [6-FAM]-CGTTAGATAGGATTCTTGGCTCGGGGTTT,
KCNQ1-ex8-R: GATAGGGTTACTTCTGCCTCTGCTTCTGCT.
After an initial denaturation step at 95°C for 9 min and before a final extension step at 72°C
for 5 min, 22 to 24 runs of the following cycle were performed: 95°C, 30 s; 57°C, 40 s; 72°C,
1 min. Amplified DNA fragments were then separated on an ABI 3730 sequencer (Applied
Biosystems), and the resulting fluorescence profiles were analyzed with Genemapper
version 3.1 software (Applied Biosystems).