High-Throughput TAIL-PCR as a Tool to Identify DNA Flanking Insertions
Thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) is a fast and efficient method to amplify unknown sequences adjacent to known insertion sites in Arabidopsis. Nested, insertion-specific primers are used together with arbitrary degenerate primers (AD primers), which are designed to differ in their annealing temperatures. Alternating cycles of high and low annealing temperature yield specific products bordered by an insertion-specific primer on one side and an AD primer on the other. Further specifity is obtained through subsequent rounds of TAIL-PCR, using nested insertion-specific primers. The increasing availability of whole genome sequences renders TAIL-PCR an attractive tool to easily identify insertion sites in large genome tagging populations through the direct sequencing of TAIL-PCR products. For large-scale functional genomics approaches, it is desirable to obtain flanking sequences for each individual in the population in a fast and cost-effective manner. In this chapter, we describe a TAIL-PCR method amenable for high-throughput production (HT-TAIL-PCR) in Arabidopsis (1). Based on this protocol, HT-TAIL-PCR may be easily adapted for other organisms.
Key WordsTAIL-PCR HT-TAIL-PCR T-DNA transposon Arabidopsis high-throughput reverse genetics tagging population knock-out