摘要
目的 探讨长片段PCR(LD-PCR)扩增特点,并将它用于对全长HIV DNA的研究。方法 应用LTR(U5)/LTR(R),CL-NSC/CL-NEF和gagA1/gagA2等不同引物,^(32)P标记gag、pol、nef和tat等为片段探针,对克隆有HIV-1完整基因片段、部分基因片段的质粒和HIV感染者外周血单核细胞(PBMC)中的HIV-1 DNA进行扩增。结果 LD-PCR对0.4～9kb的一系列标本扩增都很满意,它的扩增效率与DNA分子片段的大小成反比,在混合竞争扩增中,短片段DNA浓度较低时主要扩增长片段标本,增加短片段浓度导致减少长片段DNA的扩增。同时发现在HIV感染者中存在大量大小不一、范围较广的缺失HIV-1基因片段。结论 LD-PCR能混合扩增不同大小片段标本,特别是对大片段标本的扩增具有独特的优越性,是普通PCR无法解决的。
To study the amplification characteristics of long distance PCR (LD - PCR) and use them for amplifying full length HIV DNA. Methods The plasmids which were inserted complete, deleted HIV - 1 genomes and the HIV DNA from PBMC of HIV infected persons were amplified by primers LTR(U5)/LTR(R), CL - NSC/CL-NEF, gagAl/gagA2 and oligonucleotide 32P probe gag, pol, nef, tat, etc. Results LD - PCR was successful to amplify a series of templates sized from 0.4 kb to 9 kb, which efficiency was inversely proportional to DNA size of template. The long templates were mainly amplified when the concentration of the short templates was low in the co - amplification so that the increased concentration of short templates resulted in a reduced amplification products of the long templates. Meanwhile, the defected HIV - 1 genomes of variable size were extensively detected in PBMC of HIV infected persons. Conclusion The significance, which was observed in LD - PCR co - amplification of differently sized templates and in particular in the amplification of long templates, therefore, makes it be one of method choices in attempts to co - amplify differently size templates.
出处
《中国性病艾滋病防治》
2000年第1期1-4,共4页
Chinese Journal of Std & Aids Prevention and Control
