Concentrated rrs gene amplicons
of 277 samples were sequenced at Functional Biosciences (Madison, WI, USA) by using primers AB or CD.
The Roche 454-fusion target-specific primers, including the 454 adaptors and multiplex identifiers (MIDs) 2, 3, and 7, were designed to cover all exons of interest (Table 1; Figure 1, A) in 35 polymerase chain reaction (PCR) amplicons
. For each DNA sample, separate amplicon
PCR amplifications were performed using the FastStart High Fidelity PCR System (Roche) with 10-ng genomic DNA per reaction under touchdown thermal-cycling conditions.
Novel pooling strategies allow a minimal usage of molecular barcodes, leading to a cost reduction of 70% per sequenced amplicon
. Here we show the feasibility of using randomly Sanger sequencing-optimized PCR amplicons
in a generic and automated manner in ISS.
The Access Array Target-Specific Primers increase the number of amplicons
per sample by 1UX compared to a traditional solution.
are so called gene shuttles consisting of empty virus shells containing no viral genes.
PCR (and RT-PCR) are by their very nature reactions dependent on free diffusion of reaction components (reagents, amplicons
, and even to some extent, template).
After removing 6 genes from the data analysis because they each included amplicons
with inferior performance in library preparation or sequencing, we established performance criteria for mutational analysis of 42 genes with full or partial coverage for 157 exons.
We used a modification of COLD-PCR, fast, temperature-tolerant (fast-TT)-COLD-PCR (22) that enables mutation enrichment in amplicons
having distinct melting temperatures by use of a single PCR program.
The sample preparation, DNA extraction, PCR setup, electrophoretic separation, and sequencing of amplicons
are described elsewhere (6,9); however, we used 0.20 [micro]mol/L of each primer, an annealing temperature of 54[degrees]C, and 45 amplification cycles.
The subsequent high temperature allows the start of amplification by denaturing target strands for primer access (and in most cases, activating a "hot start" polymerase), and also acts to permanently inactivate the ung so it can't interfere with the production of new, uracil-containing amplicons
Approaches that can be used to circumvent this limitation are (a) amplicons
can be designed with specific [T.sub.m] values that instruments are capable of achieving to within [+ or -] 0.2[degrees]C and (b) amplicon
[T.sub.m] and the associated [T.sub.c] can be altered with the addition of reagents, e.g., DMSO, that increase/decrease the [T.sub.m] of the DNA duplexes to match denaturation temperatures that a PCR machine can achieve.