reverse primer (rp) PCR; thermal asymmetric interlaced (TAIL)-PCR; transferred DNA (T-DNA) repeats
한국국제농업개발학회지, v.24, no.5, pp.591 - 597
The repetitive integration of transgenes in transgenic tomatoes was investigated. Several independent transgenic lines of tomato were screened for possible T-DNA repeats using reverse primer PCR (rpPCR), which utilizes primer pairs oriented in opposite directions. The rpPCR results showed that the binding sites for primers 3 and 4 were missed and were truncated at T-DNA copies in the 3 transgenic lines, including T0-21, -26, and -34. To assess the distribution of inserts, 52 flanking sequences were amplified by thermal asymmetric interlaced (TAIL)-PCR. In the nucleotide sequence analysis results of 22 flanking regions, two showed higher than 90% homologys to centromeric region of chromosome 12, one flanking sequence showed 98% homology to tomato clone 135006R, and five flanking sequences showed 97% homology to tobacco chloroplast genome DNA. Twelve of the 22 flanking sequences showed various vector sequences and thirty flanking sequences showed no significant similarity to any released tomato genomic regions.