Wang Jianping P, Bughrara Suleiman S
Department of Crop and Soil Sciences, 286 PSSB, East Lansing, MI 48824, USA.
Mol Biotechnol. 2005 Mar;29(3):211-20. doi: 10.1385/MB:29:3:211.
In cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis, it is critical to choose a suitable pair of restriction enzymes for tagging sites in cDNA for amplification. Possibility of production of chimeric fragments from cDNA-AFLP analysis remains to be researched. The objectives of this study were to detect an efficient restriction enzyme combination for cDNA-AFLP analysis when Festuca species was used as template, and to evaluate the identity of transcript-derived fragments (TDFs) from cDNA-AFLP analysis. We found that NspI coupled TaqI was a pair of highly efficient enzymes by generating a much higher number of TDFs than the commonly used EcoRI and TaqI. This was the first study to apply NspI for AFLP analysis, prompting that this enzyme may have valuable application potential for other species. The identity of TDF was evaluated by sequencing a TDF and comparing it with the sequence of the template cDNA. The result showed that the chimeric fragments derived from ligation between digested fragments was generated and could not be eliminated by increasing adapter concentration. Although the existence of chimeric fragments should be carefully considered, the unexpected sequence in the chimeric TDF may not seriously influence the sequencing and BLAST searching analyses.
在cDNA扩增片段长度多态性（cDNA-AFLP）分析中，选择合适的一对限制性内切酶用于标记cDNA中的扩增位点至关重要。cDNA-AFLP分析产生嵌合片段的可能性仍有待研究。本研究的目的是在以羊茅属植物为模板时，检测用于cDNA-AFLP分析的高效限制性内切酶组合，并评估cDNA-AFLP分析中转录本衍生片段（TDF）的一致性。我们发现，与常用的EcoRI和TaqI相比，NspI与TaqI联用能产生更多的TDF，是一对高效的酶。这是首次将NspI应用于AFLP分析的研究，表明该酶可能对其他物种具有重要的应用潜力。通过对一个TDF进行测序并与模板cDNA序列进行比较来评估TDF的一致性。结果表明，消化片段之间连接产生的嵌合片段无法通过增加接头浓度来消除。虽然应谨慎考虑嵌合片段的存在，但嵌合TDF中的意外序列可能不会严重影响测序和BLAST搜索分析。
