The target regions were PCR-amplified from human genomic DNA using primers that incorporate the T7 [5′-CAG TAA TAC GAC TCA CTA TAG GGA GA] promoter sequence. For each target region, two sets of primers were designed to incorporate the T7 promoter sequence either to the forward or to the reverse strand. The following PCR primers were used for uniplex and multiplex reactions. Primer sequences are provided with the T7 promoter tag:

MP1_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTGAGCTATTGCGAGAATAAGGAGATG

MP1_10_REV AGGAAGAGAGCGTGTTTGCTGTGCTTGATTG

MP1_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTCGTGTTTGCTGTGCTTGATTG

MP1_10_FOR AGGAAGAGAGGAGCTATTGCGAGAATAAGGAGATG

MP2_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTCAAAATAACCAACAACCTCTTCCAG

MP2_10_REV AGGAAGAGAGGCAGAGCTCACAAGGATGGTTAC

MP2_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTGCAGAGCTCACAAGGATGGTTAC

MP2_10_FOR AGGAAGAGAGCAAAATAACCAACAACCTCTTCCAG

MP3_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTGAAGCTCAAGTTTAAAGAAGCGTTG

MP3_10_REV AGGAAGAGAGAGCTGATTCCCCTTCAAGACTATTT

MP3_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTAGCTGATTCCCCTTCAAGACTATTT

MP3_10_FOR AGGAAGAGAGGAAGCTCAAGTTTAAAGAAGCGTTG

The following PCR primer pairs were used for the CFTR multiplex of exon 10, 21 and 24:

CFTR_ex10_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTTCAGTTTTCCTGGATTATGC

CFTR_ex10_10MER_REV AGGAAGAGAGTTGGCATGCTTTGATGACGC

CFTR_ex10_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTTTGGCATGCTTTGATGACGC

CFTR_ex10_10MER_FOR AGGAAGAGAGTCAGTTTTCCTGGATTATGC

CFTR_EX21_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTGAGGTTCATTTACGTCTTTTGTG

CFTR_EX21_10MER_REV AGGAAGAGAGCATAAAAGTTAAAAAGATGATAAGACTTAC

CFTR_EX21_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTCATAAAAGTTAAAAAGATGATAAGACTTAC

CFTR_EX21_10MER_FOR AGGAAGAGAGGAGGTTCATTTACGTCTTTTGTG

CFTR_ex24_T7_FOR CAGTAATACGACTCACTATAGGGAGAAGGCTTTTCTTCTTCTTTTCTTTTTTGCTATAG

CFTR_ex24_10MER_REV AGGAAGAGAGCCCTTTCAAAATCATTTCAGTTA

CFTR_ex24_T7_REV CAGTAATACGACTCACTATAGGGAGAAGGCTCCCTTTCAAAATCATTTCAGTTA

CFTR_ex24_10MER_FOR AGGAAGAGAGTTTCTTCTTCTTTTCTTTTTTGCTATAG

The PCR reactions were carried out in a total volume of 5 μl using 1 pmol of each primer, 40 μM dNTP, 0.1 U Hot Star Taq DNA polymerase (Qiagen), 1.5 mM MgCl2 and buffer supplied with the enzyme (final concentration 1×). The reaction mix was pre-activated for 15 min at 95°C. The reactions were amplified in 45 cycles of 95°C for 20 s, 62°C for 30 s and 72°C for 30 s followed by 72°C for 3 min. Unincorporated dNTPs were dephosphorylated by adding 1.7 μl H2O and 0.3 U Shrimp Alkaline Phosphatase. The reaction was incubated at 37°C for 20 min.
Typically, 2 μl of the PCR reaction was directly used as template in a 4-μl transcription reaction. Twenty units of T7 R&DNA polymerase (Epicentre, Madison, WI) were used to incorporate either dCTP or dTTP in the transcripts. Ribonucleotides were used at 1 mM and the dNTP substrate at 2.5 mM; other components in the reaction were as recommended by the supplier. Following the in vitro transcription, RNase was added to cleave the in vitro transcript. The mixture was then further diluted with H2O to a final volume of 27 μl. Conditioning of the phosphate backbone prior to MALDI-TOF MS was achieved by the addition of 6 mg CLEAN Resin (Sequenom Inc., San Diego, CA). Further experimental details have been described elsewhere (1 (link),2 (link)).