Molecular Biotechnology Research Laboratory, Boston University, Boston, MA, USA.
J Clin Lab Anal. 2011;25(4):259-88. doi: 10.1002/jcla.20470.
The many techniques used to diagnose the Factor V Leiden (FVL) mutation, the most common hereditary hypercoagulation disorder in Eurasians, and the most frequently requested genetic test reflect the evolving strategies in protein and DNA diagnosis.
Here, molecular methods to diagnose the FVL mutation are discussed.
Protein-based detection assays include the conventional functional activated protein C resistance coagulation test and the recently reported antibody-mediated sensor detection; and DNA-based assays include approaches that use electrophoretic fractionation e.g., restriction fragment length polymorphism, denaturing gradient gel electrophoresis, and single-stranded conformational PCR analysis, DNA hybridization (e.g., microarrays), DNA polymerase-based assays, e.g., extension reactions, fluorescence polarization template-directed dye-terminator incorporation, PCR assays (e.g., amplification-refractory mutation system, melting curve analysis using real-time quantitative PCR, and helicase-dependent amplification), DNA sequencing (e.g., direct sequencing, pyrosequencing), cleavase-based Invader assay and ligase-based assays (e.g., oligonucleotide ligation assay and ligase-mediated rolling circle amplification).
The method chosen by a laboratory to diagnose FVL not only depends on the available technical expertise and equipment, but also the type, variety, and extent of other genetic disorders being diagnosed.
用于诊断因子 V 莱顿（FVL）突变的多种技术，这是欧亚人最常见的遗传性高凝状态，也是最常要求进行的基因检测，反映了蛋白质和 DNA 诊断策略的不断发展。
本文讨论了用于诊断 FVL 突变的分子方法。
基于蛋白质的检测方法包括传统的功能性激活蛋白 C 抵抗凝血试验和最近报道的抗体介导的传感器检测；基于 DNA 的方法包括使用电泳分馏的方法，例如限制片段长度多态性、变性梯度凝胶电泳和单链构象 PCR 分析、DNA 杂交（例如微阵列）、基于 DNA 聚合酶的方法，例如延伸反应、荧光偏振模板指导染料终止物掺入、PCR 检测（例如扩增不可抑制突变系统、实时定量 PCR 的熔解曲线分析、和依赖解旋酶的扩增）、DNA 测序（例如直接测序、焦磷酸测序）、基于切割酶的 Invader 检测和基于连接酶的检测（例如寡核苷酸连接检测和连接酶介导的滚环扩增）。
实验室选择用于诊断 FVL 的方法不仅取决于可用的技术专业知识和设备，还取决于正在诊断的其他遗传疾病的类型、种类和程度。
