A forward primer bearing the desired mutation six base pairs from its 5′ end anneals immediately downstream from a reverse primer with wild-type sequence (A).
In addition, sequencing success was low from the reverse primer direction.
Although sequence information for the central portion of the gene is missing, using a forward primer from contig 23485908 and a reverse primer from contig 83285534, a ∼1.5 kb fragment is amplified from genomic DNA while multiple bands are observed from cDNA confirming that these two contigs are contiguous (1.PY_PFB0505c in Table 6 and Fig. 3).
To evaluate the quality and consistency of cDNA from this complex reverse transcription reaction, we compared the expression values resulting from 8 smaller reverse primer pools (each containing 94 to 96 primers) to those from the single pool with all of the primers combined.
Six primer pairs were designed within the VPaI-1 region, two primer pairs encompassing each of the VPaI-1 flanking core chromosomal genes, VP0379 and VP0404, and a primer pair comprised of a forward primer from VP0379 and a reverse primer designed from VP0404 (Table 3).
Myzus persicae cultures are genotyped at six loci: M40 (forward primer labeled with VIC, M40-for-VIC) and M63 (M63-for-6-FAM) from [ 58]; myz2 (reverse primer labeled with PET, myz2 rev-PET), myz2 rev-PETor-VIC), myz95 (myz9-for-VICD), and S17b (S17b-for-NED) fromyz259].
To do this, we used the program Oligo 6 [ 27] to design a set of primers against the NC_001422.1 Genbank reference sequence, that gave three fragments of approximately 200 bp, 400 bp and 600 bp from a common reverse primer and that had roughly equal Tm and priming efficiencies.
The length from forward and reverse primer to TALEN-702 cleavage site is 883 and 611 bp, respectively, so the PCR product can be digested into two fragments (883 and 611 bp) only if NHEJ events occur at TALEN-702 cleavage site.
The sequence was further confirmed using a forward primer from newly identified sequence and a reverse primer from known sequence.
Upon observing equal amplification (i.e., from all animals) of fragments located on both sides of the allele-specific amplicon, different combinations of primer pairs, consisting of a forward primer from the upstream region and a reverse primer from the downstream region, were used to amplify across the breakpoint.
