Abstract:

【Objective】 This study aimed at establishing a protocol to identify unknown target sequences adjacent to T-DNA borders. 【Method】 DNA tagging by T-DNA insertions has become an important approach for study of functional genomics in plants. To identify the genes tagged by T-DNA insertions, a novel and efficient procedure，named as annealing control thermal asymmetric interlaced PCR (Actail-PCR), was developed to isolate genomic sequences flanking the insertion tags. In this procedure, four nested sequence-specific primers from T-DNA were utilized. The other side primer was a annealing control primer (ACP), which comprises a tripartite structure with a polydeoxyinosine (poly (dI)) linker between the 3' end shorter arbitrary degenerated primer sequence (AD) and the 5' end nontarget universal sequence. 【Result】 Annealing control primers were designed for Actail-PCR instead of shorter arbitrary degenerated primers of TAIL-PCR. At 40℃ low stringency, PCR cycle was conducted to create one or more annealing sites for the AD primer along the target sequence like TAIL-PCR, and then, target products were preferentially amplified over 5' end nontarget universal primer and nested sequence-specific primers from T-DNA at 65℃ (after 10 cycles, annealing temperature droped to 58℃) high-stringency, so the efficiency and specificity of PCR amplification were greatly improved. 【Conclusion】 An novel procedure for isolation of unknown target sequences adjacent to T-DNA in the rice has been established. Actail-PCR is more efficient and useful for identification of the genes tagged by T-DNA insertions

Key words: T-DNA, flank sequence, TAIL-PCR, Actail-PCR