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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。
OBJECTIVE: To investigate the characteristics of long-length PCR (LD-PCR) amplification and to use it for the study of full-length HIV DNA. METHODS: Different primers, such as LTR (U5) / LTR (R), CL-NSC / CL-NEF and gagA1 / gagA2, were used to detect gag, pol, nef and tat fragments by ~ (32) -1 complete gene fragment, a partial gene fragment and HIV-1 DNA in HIV-infected peripheral blood mononuclear cells (PBMCs). Results LD-PCR was very satisfactory for amplification of a series of samples from 0.4 to 9 kb. The amplification efficiency of LD-PCR was inversely proportional to the size of DNA fragments. In mixed competition amplification, Fragment samples, increasing the short fragment concentration lead to the reduction of long fragment DNA amplification. At the same time, it was found that a large number of HIV-1 gene fragments lacking a wide range of sizes exist in HIV-infected persons. Conclusion LD-PCR can mix and amplify samples of different sizes, especially for the amplification of large fragments, which can not be solved by ordinary PCR.