摘要
下一代测序技术目前已经应用于微生物、人类、动物、植物等的基因组分析。样品制备是开展大规模测序的必要前提和测序成功的根本保证。对大规模测序造成干扰的主要因素有:polyA干扰测序信号及高丰度基因对低丰度基因的掩盖等。文章以堇菜(Viola verecumda A.Gray)叶片为试材,提取总RNA,合成双链cDNA,利用DSN核酸酶对双链cDNA进行均一化处理,并对双链cDNA polyA进行了切除,将处理后的cDNA进行了TA克隆,挑取100个克隆随机测序。结果表明,未处理的cDNA样本测序有15个克隆由于polyA的存在而影响了附近碱基的正确阅读,独立克隆只有62个,而处理后的cDNA样本经测序未发现polyA,独立克隆有94个。序列分析发现,经过处理的cDNA样本随机测序有两个克隆是经MALDI-TOF检测在样本中有蛋白质峰,而基因克隆一直没有分离到的序列。以处理后的cDNA样本为模板扩增2个已知表达丰度差异较大的基因显示,处理后这2个基因的PCR扩增产量差异明显减小。这些结果表明polyA切除、DSN核酸酶处理的cDNA样本完全满足大规模测序、寻找新基因的要求。
Next generation sequencing has already been used for genomic analysis of microorganis, human being, animals and plants. Sample preparation is prerequisite and most important for large-scale sequencing. There are two major interferences for large-scale sequencing, polyA and abundant genes＇ concealment for rare genes. In order to solve these problems, we used total RNA extracted from violaceae leaves to produce double stranded cDNA. DSN nuclease was used to treat the ds cDNA prior to removing the polyA. Randomly sequencing 100 clones of the treated cDNA showed that there were 94 independent clones in the treated sample, and the sequences did not contained polyA. However, only 62 independent clones were found in the untreated sample, and 15 of the sequencing files were affected by polyA. By randomly sequencing of the treated cDNA, we also found two clones encoded two interested genes. We failed to isolate these genes although the protein mass peaks of them had been found in the MALDI-TOF trace. Furthermore, we designed primers from two known genes with different expression abundances. The PCR yields were approaching similar using the treated cDNAs as templates. These results showed that, removal of the polyA and enrichment of rare genes with DSN can meet the requirements of large-scale sequencing and discovery of new genes.
出处
《遗传》
CAS
CSCD
北大核心
2010年第9期974-977,共4页
Hereditas（Beijing)
基金
国家留学基金项目(编号:2007106650)
国家自然科学基金项目(编号:30771762)
浙江农林大学青年教师创新团队项目(编号:2009RC02)
国家科技支撑计划子课题(编号:2008BAD92B08-6)
林业科技支撑计划子课题(编号:2006BAD03A15)
浙江省自然科学基金项目(编号:Y305222)资助
关键词
CDNA
下一代测序
堇菜
cDNA
next generation sequencing
Viola verecumda A.Gray
