Primers were designed to correct these errors by PCR: "Correc65": CATCCCTCCTCCCT TTAAATTCAGATCCC "Correc74": GGCGTGAAAATG TGGACTGGCGGCGC "Correc488": ACAGGTCTTGGCA GAAGGAGGTGCAAG The construct was then corrected using Stratagene's multi-site directed mutagenesis kit (Agilent Technologies, Cheshire, UK) according to the manufacturer's instructions and confirmed by sequencing.
To correct for primer efficiency, each primer pair was also used in the same PCR run to amplify varying amount of G-block DNA (IDT, Coralville, IA).
The assay is quantitative, having used a complete synthetic repertoire of TCRs to establish an amplification baseline and adjust the assay chemistry to correct for primer bias.
As two examples for STRPs on chromosome 1 in Set 12: GATA26G09N indicates that one of the original primers for GATA26G09 was changed to correct a sequencing error without change in allele sizes, and GGAA3A07Z indicates that one of the primers for GGAA3A07 was shifted along the chromosome resulting in different allele sizes.
PCR bands were sequenced with Gal4 reverse primer to confirm correct reading frame of the fusion mRNA.
Each sequence was edited to correct sequencing ambiguities, remove primer sequence and exclude chimeric clones.
To calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies, a standard curve was constructed.
To calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies for each gene a standard curve was constructed.
A standard curve, derived from known serial dilutions of RA synovial tissue, was constructed to calculate arbitrary values of mRNA levels and to correct for differences in primer efficiencies.
Five clones of each were selected and sequenced on the ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) using the vector primers available from the cloning kit (Invitrogen) as sequencing primers, to ensure correct insertion of the amplicons into the plasmids.
