The concentration of these fragments of 400 to 600 bp was in the range of 50 to 600 pg µL−1, in agreement with our measurements at different stages of the PCR amplification (Table 2).
For four of the five samples, we could detect two alleles with lengths consistent with the prediction based on Sanger sequencing and agarose gel electrophoresis after 45 cycles of amplification (Table 2 and Fig. 6A).
The size and specificity of each amplicon was confirmed using agarose gel electrophoresis and melting curve analysis (Fig. 1 and Supplementary Figure 2), and the no template controls (NTCs) demonstrated non-detectable levels of amplification (Table 1 and Supplementary Figure 2).
A battery of computational models have been developed to address false-positives due to nonlinear amplification, false-negative allelic drop-out due to amplification bias, non-uniform coverage, and noise that arises during single-cell genome or transcript amplification (Table 2) [40].
Interestingly, this result seemed to correlate with MYCN amplification (Table 1).
Primers P1 and P2 were used for ldhL amplification and primers P3 and P4 were used for ldhD amplification (Table 4).
Core region was amplified by RT-nested PCR with primers 939P-C4N for the first round and combinations of C5P with C4N, C6N or C9N, in order to maximize the success in amplification (Table 1).
Couples of primers were designed on different exons using Primer 3 software to avoid DNA amplification (Table S1) and for each couple of primers, all qPCRs displayed efficiency between 90% and 110%.
Here, we studied the copy number and location of region B in these 25 strains, together with three additional wine yeast strains or derivatives (59A, V5 and N96), through a combination of PFGE, Southern blotting and PCR amplification (Table S1).
574-GFP (encoded by p574-c20, GU994008) was constructed by gene assembly using oligonucleotides of ∼50 nt overlapping by 20 30 bases, and external 5'-biotinylated primers otb141 and otb151 for amplification (Table S2 and Table S3).
Samples were segregated based on CCNE1 copy number level as assessed by qPCR (above) using a log2 copy number ratio cut-off of ≥0.5 (∼3 copies) for gain and ≥2 (∼8 copies) for amplification (Table 1).
