Besides, bands of same size may not come from the same part of the chromosome, change in one restriction site can result in more than one band change, and inability to differentiate isolates to the same degree that can be achieved by whole genome sequencing (WGS) are other key restraining factors.
In order to clone the fragment containing the [beta]-globin gene promoter in a pBluescript II SK (pSK) vector, [beta]_KpnI and [beta]_XhoI primers, engineered to contain both KpnI and XhoI restriction site, respectively, were used.
1) was produced using oligonucleotide primers p903SalI (5'GTCGACATAAG TAGGAAATTAAAGTCCAGTAAGGTTACTGGCATTTCT [This oligonucleotide primer was appended with a SalI restriction site (underlined) and the introduced late gene polyhedrin core promoter sequence, ATAAG (bold)]), and p738 (5'ACGAGCT GTGAACT CACCAAGAAT CCAACGTT) with cDNA being generated using oligonucleotide primer p738.
Therefore, longer fragments affected by ADO could be misinterpreted as alleles without a mutation in the restriction site. This problem can be avoided with ddRAD, for which two restriction enzymes are utilized (Davey et al., 2013).
Oligonucleotides used to amplify CLCuKoV promoter were P1 (5' GTTGACTAAAATTGAATCACC-3') as forward with the addition of SacI restriction site and P2 (5'- CAAACGCATACTTAGCAACG-3') as reverse primer with the addition of SalI restriction site.