However, recently developed novel methods for universal sequence
dependent (USD) NAT are superior to conventional approaches in that they employ standardized fluorogenic reagents that provide cost-efficient detection of various targets.
This low-cost method of genotyping involves three primers: the forward primer of each locus which contains the M13 (-21) universal sequence
(18bp) at the 5' end (5'- TCTAAA ACGACGGGCACT 3'), a locus-specific reverse primer and a third fluorescentlylabeled HEX-M13 (-21) universal primer: HEX-TGTAAA ACGACGGGCACT-3' (5-HEX=hexachloro-6-carboxy-fluorescine).
difficile amplification compared to universal sequence
amplification is automatically calculated by the Opticon 3.1 software of the Chromo 4 real-time detector, using the formula [2.sup.[DELTA]Ct].
To amplify translocation-specific transcripts present in leukemia cells, primers contained sequences specific to those flanking the junction of fusion genes as well as the T7 and SP6 universal sequence
The universal sequences
of primers used for the forward primer was T7 and the reverse primer were slightly modified RP2 or M13R.
Because the length bias of the shotgun sequencing reads was suspected to derive from the PCR, one could potentially ligate universal adaptors to the ends of plasma DNA and then perform PCR amplification against the universal sequences
to enrich the fetal DNA fraction.
Although only 12 universal sequences
were used for this assay, empirical validation of the full 100-plex array established its potential as a highly specific sorting tool for multiplex analysis of up to 50 biallelic SNPs or 100 analytes.