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在对中华绒鳌蟹(Eriocheirjaponicasinensis)(又称河蟹)核糖体DNA内转录间隔区1(ITS1)进行研究时,由于在软甲亚纲中还没有已知序列和5.8SrDNA序列的报道,所以在设计引物时,主要参考了其它节肢动物的序列。用此引物对河蟹的ITS1进行扩增,发现其个体内ITS1如同其他一些无脊椎动物一样,存在其大小变异。为了进一步确证这一发现,首先将得到的所有扩增产物进行测序,然后再扩增出河蟹的整个ITS区并测序。通过所得序列的比较,发现河蟹个体内ITS1的大小变异是由引物错配而引起的赝相。为了检验这一结论的可靠性,该文在测出河蟹5.8SrDNA序列的基础上,重新设计了引物,并再次扩增了河蟹的ITS1,然后对产物进行测序,最后再同上述结果进行比较;同时在改变扩增条件的情况下用原引物重新扩增ITS1,并检验其结果。最终证实河蟹个体内ITS1的大小变异是由引物错配而引起的赝相。该文同时还报道了中华绒螯蟹ITS1的序列。
In the study of ribosomal DNA transcribed spacer 1 (ITS1) in Eriocheirjaponicasinensis (also known as crab), since there is no known sequence and 5.8S rDNA sequence reported in the subclass, Primer design, the main reference to other arthropod sequences. Using this primer to amplify ITS1 of crabs, we found that ITS1, as some other invertebrates, has its size variation in ITS1. To further confirm this finding, all amplified products were first sequenced, and then the entire ITS region of crabs was amplified and sequenced. Through the comparison of the obtained sequences, it was found that the variation of the ITS1 size in crabs was caused by the mismatch of primers. In order to test the reliability of this conclusion, based on the 5.8SrDNA sequence of crabs, the primers were designed and the ITS1 of crabs was amplified again. Then the products were sequenced and compared with the above results. At the same time, the amplification conditions were changed with the original primer ITS1 re-amplification, and test the results. Finally, it was confirmed that the variation of ITS1 in crab individuals was caused by the mismatch of primers. The article also reported the ITS1 sequence of Chinese mitten crab.