摘要
针对mRNA差异显示技术中的假阳性高和放射性污染等缺陷,文章从改进引物设计、应用非放射性标记、优化PCR参数、假阳性鉴定等方面进行了综述。1采用4种兼并或单碱基引物作锚定引物;用特异和长序列(≥20bp)专一性引物作为随机引物,可增加特异性;在锚定引物之前加上了一段T7启动子序列,在随机引物前加上一段M13重复序列,可使DDRT—PCR的体系的扩增特异性更一致。2针对放射性同位素检测法的缺点,文章提出并比较了几种常用的非放射性同位素标记方法。3保证起始(模板)mRNA质量和浓度,降低dNTP浓度,选择最适的反转录和PCR退火温度,可优化PCR参数。4用两种不同的反转录酶进行反转录平行实验,很容易区分出RNA制备过程中造成的假阳性,目前假阳性鉴定大多还是以Northern印迹为基础,再作些适当的改进。
Aim at flaws of mRNA differentially display technology which higher false-positive and radioactive pollution,some methods is summarized that such as improve primer design,application of non-radioactive marker,optimization of parameter of PCR, false-positive identification. First, use four type degenerate or single -nucleotide primers for anchor- primer; peculiar and longer(≥20bp)specificity primer for random-primer, which can increase specificity ; while a T_7 promoter sequence and a M_(13) reverse sequence were added on both sides of anchor- primer and random- primer ,then DDRT-PCR system have better identical than before process in amplification specificity. Second, aim at flaws of method radioactive marker, same methods of non-radioactive marker were raised and compared. Third, assure quality and amount of mRNA, reduce amount of dNTP ,select of suitable temperature of inverse transcription and PCR renaturation(the primers to anneal to the template DNA strands),the methods can optimize parameter of PCR. Forth, make inverse transcription parallel experiment with difference inverse transcriptase, it is easy to distinguish from false-positive process in RNA process in experiment, today, Northern blot is better method on false-positive of identification,some peoples who have result well by improved correctly.
出处
《西北农业学报》
CAS
CSCD
北大核心
2005年第2期9-12,43,共5页
Acta Agriculturae Boreali-occidentalis Sinica
基金
湖南省杰出青年基金(03JJY1004)
中国科学院知识创新工程重要方向项目(KZCX3-SW-434)。
