Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction.
Increased sample capacity for genotyping and expression profiling by kinetic polymerase chain reaction.
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通过动态聚合酶链反应增加基因分型和表达谱的样品容量。
DOI：
10.1016/j.ab.2004.02.018
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Holland,MichaelJ
中科院分区：
文献类型：
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作者：
Watson,RobertM;Griaznova,OlgaI;Long,ChristopherM;Holland,MichaelJ
我们制造并评估了具有768个反应容量的高通量动力学热循环器，用于基于动力学聚合酶链反应（kPCR）的基因分型和基于动力学逆转录(kRT)- pcr的转录物定量。该系统使用染料为基础的检测与溴化乙啶和单一的DNA聚合酶为基础的PCR或RT-PCR测定。两种最常见的遗传性血色素突变等位基因C282Y和H63D的等位基因特异性检测通过kPCR可靠地测量，每次检测使用的人类DNA模板低至10个基因组当量。转录分析对16个酵母转录本进行了分析，细胞内丰度超过4个数量级。在三种不同的仪器中，通过多个kRT-PCR检测确定的PCR周期阈值的标准偏差范围为0.11至0.97 PCR周期，并且具有可重复性、转录特异性和仪器独立性。通过kRT-PCR评估了sin3、gal11和snf2敲除突变对385个酵母基因表达的影响，并与已发表的高密度寡核苷酸阵列和/或微阵列分析snf2和sin3的结果进行了比较。768个反应动力学热循环仪，每个具有每年超过50万次分析的能力，非常适合基因组学应用，如单核苷酸多态性/疾病关联研究和全基因组转录分析，这些都需要高灵敏度和准确性。
We fabricated and evaluated high-throughput kinetic thermal cyclers with 768-reaction capacity for kinetic polymerase chain reaction (kPCR)-based genotyping and kinetic reverse transcription (kRT)-PCR-based transcript quantitation. The system uses dye-based detection with ethidium bromide and a single DNA polymerase-based PCR or RT-PCR assay. Allele-specific detection of the two most common hereditary hemochromotosis mutant alleles, C282Y and H63D, was reliably measured by kPCR using human DNA templates as low as 10 genome equivalents per assay. Transcript profiling was performed for 16 yeast transcripts ranging in intracellular abundance over four orders of magnitude. Standard deviations of the PCR cycle threshold values determined from multiple kRT-PCR assays in three different instruments ranged from 0.11 to 0.97 PCR cycles and were reproducible, transcript specific, and instrument independent. The effects of the sin3, gal11, and snf2 knockout mutations on expression of 385 yeast genes were evaluated by kRT-PCR and compared to published values determined by high-density oligonucleotide array and/or microarray analysis for snf2 and sin3. The 768-reaction kinetic thermalcyclers, each with a capacity for more than a half million assays per year, are well suited to genomics applications such as single nucleotide polymorphism/disease association studies and genomewide transcription profiling where high sensitivity and accuracy are required.