Using Syber Green Master Mix (Applied Biosystems, Foster City, CA), amplification was performed in ABI Prism 8500 system for 40 cycles in quadruplicate.
Our combined in vitro / in vivo selection scheme was designed to foster the self-amplification of novel polymerase variants.
After purifying the PCR products by treatment with ExoSAP-It (usb Corp., Cleveland, OH) at 37°C, they were sequenced on an automated DNA sequencer (ABI Prism GA310; Applied Biosystems, Foster City, CA) with amplification primer H15915, using the BigDye Terminator Cycle Sequencing FS Ready Reaction Kit Ver. 1.1 (Applied Biosciences).
Some genomes appear to have whole-chromosome amplifications: chromosome I in AWRI796, chromosome III in Foster's O and chromosomes II, V and XV in Foster's B. Several partial chromosomal CNV amplifications hundreds of kilobases long were also identified, as were some reductions in copy number.
Synthesized cDNA was then used for real-time qPCR amplification on ABI stepone plus PCR equipment (Applied Biosystems, Foster City, CA, USA).
The SSR markers were screened for amplification and length polymorphisms using GeneMapper 3.0 software (Applied Biosystems, Foster City, CA).
PCR amplification was performed using a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).
This agrees with observations of Stumpf and Foster (Stumpf et al. 2007) and suggests that the preexisting lac amplification critical to revertant initiation is provided by increased copy number of the entire F′ lac plasmid.
PCR amplification was carried out with a PE9700 thermal cycler (Applied Biosystem Inc., Drive Foster City, USA).
Single nucleotide polymorphism (SNP) genotyping was performed with SNPlex Technology Applied Biosystemss, Foster City, CA) based on oligonucleotide ligation assay combined with multiplex PCR target amplification (http://www.appliedbiosystems.com) (14).
