代表性差异分析和消减杂交差异显示分析方法在克隆差异基因方面的灵敏度比较

Comparison of detection sensitivity of RDA and SHDD methods in cloning differential genes

目的建立目的基因差异信息标量化的配对研究体系，比较经典的代表性差异分析（RDA）方法和消减杂交差异显示分析（SHDD）方法在克隆差异基因方面的灵敏度。方法以人类乳头瘤病毒（HPV）DNA为模板行PCR扩增获得376bp和869bp两个片段，作为差异目的基因掺入人血基因组DNA，获得5组目的基因拷贝数呈梯度差异的配对研究体系，比较RDA和SHDD方法的检测灵敏度。结果应用RDA方法可克隆得到上调6倍的376bp片段和上调8倍的869bp的HPV片段，而应用SHDD方法可克隆到上调4倍的两种差异片段。结论SHDD方法由于采用了两样本差异产物问的双向均衡消减杂交，避免了经典RDA方法不均衡消减杂交导致的差异信息的丢失，在克隆较弱的差异信息，尤其是较长差异信息方面，显著优于经典RDA方法。

Objective To establish matched-pairs of DNA samples with copy-number controlled differential target genes, and to compare the detection sensitivity of typical Representational Difference Analysis （RDA） method and Subtractive Hybridization Difference Display （SHDD） method in isolating differential genes. Methods Two gene fragments （376 bp and 869 bp in length respectively） cloned by PCR using Human Papilloma Virus （HPV） DNA as template were used as differential target genes, and mixed with human genome DNA. Five matched-pairs of human genome DNA samples with gradually increased difference in copy numbers of target genes were established and RDA and SHDD methods were performed to clone differential target genes and compared their detection sensitivity. Results By using RDA method, 376 bp fragment with 6-fold difference and 869 bp fragment with 8-fold difference were cloned. However, both of these two target fragments with 4-fold difference were isolated using SHDD method. Condusion The SHDD method adopts balanced bi-directional subtractive hybridization between two sample difference products and avoids loss of differential .target genes caused by unbalanced subtraetive hybridization of RDA method, and thus outweighs RDA method in isolating target genes, especially long-length target fragments, with small difference.