Luthra Rajyalakshmi, Medeiros L Jeffrey
Department of Hematopathology, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.
Methods Mol Biol. 2006;335:135-45. doi: 10.1385/1-59745-069-3:135.
Real-time TaqMan polymerase chain reaction (PCR) assays allow quantification of the initial amount of target in a specimen, specifically, and reproducibly. The major limitation of TaqMan PCR assays is that they do not detect the size of the amplified target sequence. TaqMan PCR coupled with capillary electrophoresis is an alternative approach that can be used to circumvent this limitation. In this chapter, the utility of this approach in the identification and quantification of bcr-abl fusion transcripts produced as a result of t(9;22)(q34;q11) in chronic myelogenous leukemia is described. In this assay, abl primer labeled at its 5'-end with the fluorescent dye NED (Applied Biosystems [ABI], Foster City, CA) is incorporated into the bcr-abl fusion product during the real-time PCR. The incorporated NED fluorescent dye is then used subsequently to identify the specific fusion transcript present in a given specimen by high-resolution capillary electrophoresis and GeneScan (ABI) analysis. Knowledge of the type of fusion transcript present in a specimen is useful to rule out false-positive results and to compare clones before and after therapy.
实时TaqMan聚合酶链反应(PCR)检测能够特异性且可重复地对样本中靶标的初始量进行定量。TaqMan PCR检测的主要局限性在于它们无法检测扩增靶标序列的大小。TaqMan PCR与毛细管电泳相结合是一种可用于克服这一局限性的替代方法。在本章中,将描述这种方法在鉴定和定量慢性粒细胞白血病中因t(9;22)(q34;q11)产生的bcr-abl融合转录本方面的应用。在该检测中,在实时PCR过程中,5'-端用荧光染料NED(应用生物系统公司[ABI],加利福尼亚州福斯特城)标记的abl引物会掺入到bcr-abl融合产物中。随后,通过高分辨率毛细管电泳和基因扫描(ABI)分析,利用掺入的NED荧光染料来鉴定给定样本中存在的特定融合转录本。了解样本中存在的融合转录本类型有助于排除假阳性结果,并比较治疗前后的克隆。