D17Z1探针点杂交DNA定量研究

DNA QUANTIFICATION BASED ON D17Z1PR OBE SLOT -BLOT HYBRIDIZATION TO A HUMAN ALPHA SATEL LITE LOCUS

本研究化学合成了高等灵长类特异性α卫星寡核苷酸片段(D17Z1),经辣根过氧化物酶标记、分子杂交、化学发光法检测,建立了高等灵长类特异性DNA精确定量的方法。制备了DNA浓度梯度标准对照。对人类DNA,猴、猪、牛、羊、鸡、兔、鱼、小鼠等常见动物DNA及JM109大肠杆菌、λDNA、Ф174DNA等微生物DNA进行了定量分析。结果表明,应用该方法对人类DNA定量不受非高等灵长类动物DNA与微生物DNA的影响,可实现组分定量;灵敏度测试,可对0.12ng的人类DNA进行定量,适用于法医DNA检验定量分析。

A simple,accurate and sensitive pro cedure for quantitation of human gen omic DNA is described.The method is based on the hybridization of a HRP labelled oligonucleotide pr obe to sample DNA immobilized on nylo n membrane,followed by enhanced chem iluminescent detection and autorad iography.The probe is complementary to a primate specific alpha satellite DNA sequence on chromosome 17(D17Z1).Titration series of a human genomic DNA standard of known concentration is p repared.The quantity of DNA present in the sample extract slot is estimated by comparison with the slots produced f rom a titration series of a standard DNA sample.Human and non -human DNA have been tested and the results indi cate very high specificity to human DNA.120pg of human DNA can be detected with a 15minutes exposure.The specificity and sensitivity of this quant itation method for human DNA will be u seful for the analysis of samples that may be co ntaminated by bacterial or other non-human DNA,which often occurred in forensic evidence samples.