| High-throughput TAIL-PCR as a tool to identify DNA flanking insertions | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-29 本站论坛 |
|  | 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 specificity 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. Based on this protocol, HT-TAIL PCR may be easily adapted for other organisms.
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