Castley Alison, Higgins Melinda, Ivey John, Mamotte Cyril, Sayer David C, Christiansen Frank T
Department of Clinical Immunology, Haematology, Royal Perth Hospital, Australia.
Clin Chem. 2005 Nov;51(11):2025-30. doi: 10.1373/clinchem.2005.055327. Epub 2005 Sep 15.
As the genetic basis of many human diseases is being discovered, there is increasing need for the detection of single-nucleotide polymorphisms/mutations in medical laboratories. We describe an innovative approach that combines PCR amplification directly on whole blood and real-time detection PCR technology (WB-RTD PCR).
We compared WB-RTD PCR with the method for extracted DNA-RTD PCR for the detection of mutations in the prothrombin (n = 94), factor V Leiden (n = 49), and hemochromatosis (n = 22) genes. Mutation detection on the Roche LightCycler was based on use of fluorescence resonance energy transfer (FRET) probes and melting curve analysis. We also compared the WB-RTD PCR on the LightCycler and the ABI Prismtrade mark 7700 sequence detection system with minor groove- binding nonfluorescent quencher probes.
We obtained complete concordance between both methods in assigning genotypes. We also demonstrated that the WB-RTD PCR method can be performed on real-time PCR instruments from Applied Biosystems and the LightCycler. Omission of the need for DNA extraction and gel electrophoresis allowed substantial labor and cost savings with this method.
This approach has applications for testing other medically relevant single-nucleotide polymorphisms.
随着许多人类疾病的遗传基础被发现，医学实验室对单核苷酸多态性/突变检测的需求日益增加。我们描述了一种创新方法，该方法将直接在全血上进行的PCR扩增与实时检测PCR技术（WB-RTD PCR）相结合。
我们将WB-RTD PCR与提取DNA的RTD PCR方法进行比较，以检测凝血酶原（n = 94）、因子V Leiden（n = 49）和血色素沉着症（n = 22）基因中的突变。在罗氏LightCycler上进行的突变检测基于荧光共振能量转移（FRET）探针和熔解曲线分析。我们还比较了在LightCycler上的WB-RTD PCR和使用小沟结合非荧光淬灭探针的ABI Prism商标7700序列检测系统。
我们在确定基因型方面两种方法完全一致。我们还证明了WB-RTD PCR方法可在应用生物系统公司的实时PCR仪器和LightCycler上进行。省去DNA提取和凝胶电泳的步骤使得该方法节省了大量人力和成本。
这种方法可用于检测其他医学相关的单核苷酸多态性。
