Comparison of detection sensitivity of RDA and SHDD methods in cloning differential genes / 肿瘤研究与临床

ABSTRACT

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.Conclusion The SHDD method adopts balanced bi-directional subtractive hybridization between two sample difference products and avoids loss of differential target genes caused by unbalanced subtractive hybridization of RDA method, and thus outweighs RDA method in isolating target genes, especially long-length target fragments, with small difference.