Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.
Human genomic DNA quantitation system, H-Quant: development and validation for use in forensic casework.
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人类基因组 DNA 定量系统 H-Quant：用于法医案件的开发和验证。
DOI：
10.1111/j.1556-4029.2006.00369.x
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发表时间：
2007
影响因子：
1.6
通讯作者：
Sinha,SudhirK
中科院分区：
文献类型：
--
作者：
Shewale,JaiprakashG;Schneida,Elaine;Wilson,Jonathan;Walker,JerilynA;Batzer,MarkA;Sinha,SudhirK
人类DNA定量（H‐Quant）系统，开发用于人类鉴定，可以定量生物样品中的人类基因组DNA。该分析是基于在类人猿灵长类动物中aluyb8插入的实时扩增。人类基因组中亚家族特异性alurepeats的拷贝数相对较高，可以定量分析非常少量的人类DNA。H - Quant中存在的寡核苷酸引物是针对人类DNA和近亲类人猿的特异性引物。在实时PCR中，SYBR®Green I染料结合到由人类特异性aluyb8寡核苷酸引物合成的DNA上。在每个PCR周期结束时测量结合的SYBR®Green I染料的荧光。荧光越过所选阈值的周期与样品中可扩增DNA的数量相关。H - Quant系统的最小灵敏度为7.6 pg/μL。在H‐Quant实验中产生的扩增子为216 bp，与常见的可扩增短串联重复序列（STR）扩增子的范围相同。这种大小的扩增子可以对可扩增的DNA进行定量，而不是对较小尺寸的降解或不可扩增的DNA进行定量。根据法医DNA检测实验室的质量保证标准，对7500实时PCR系统进行了开发和验证研究。
The human DNA quantification (H‐Quant) system, developed for use in human identification, enables quantitation of human genomic DNA in biological samples. The assay is based on real‐time amplification ofAluYb8 insertions in hominoid primates. The relatively high copy number of subfamily‐specificAlurepeats in the human genome enables quantification of very small amounts of human DNA. The oligonucleotide primers present in H‐Quant are specific for human DNA and closely related great apes. During the real‐time PCR, the SYBR®Green I dye binds to the DNA that is synthesized by the human‐specificAluYb8 oligonucleotide primers. The fluorescence of the bound SYBR®Green I dye is measured at the end of each PCR cycle. The cycle at which the fluorescence crosses the chosen threshold correlates to the quantity of amplifiable DNA in that sample. The minimal sensitivity of the H‐Quant system is 7.6 pg/μL of human DNA. The amplicon generated in the H‐Quant assay is 216 bp, which is within the same range of the common amplifiable short tandem repeat (STR) amplicons. This size amplicon enables quantitation of amplifiable DNA as opposed to a quantitation of degraded or nonamplifiable DNA of smaller sizes. Development and validation studies were performed on the 7500 real‐time PCR system following the Quality Assurance Standards for Forensic DNA Testing Laboratories.