Ramasamy I, Brisco M, Morley A
Department of Biochemistry, Repatriation General Hospital, Daw Park, SA.
J Clin Pathol. 1992 Sep;45(9):770-5. doi: 10.1136/jcp.45.9.770.
To develop a simple, optimised, polymerase chain reaction (PCR) based method for detecting the rearranged immunoglobulin heavy chain (IgH).
Using as primers oligonucleotides (Fr2A, Fr2B) homologous to the conserved sequences to the framework II region and the joining (JH) region, 25 patients with B cell lymphoproliferative disorders, previously characterised by Southern blotting, and three patients with light chain myeloma were studied.
The PCR product from a polyclonal B cell population showed a broad band when analysed on a 3% agarose gel; DNA from B cell lines and B lymphoproliferative disorders showed a discrete band. Specificity of the amplification was confirmed by cloning and sequencing the amplified product as well as by Southern blotting with an internal probe homologous to the framework 3 region. Primers Fr2A and Fr2B detected monoclonality in three patients with light chain myeloma, while primers directed against the FrIII region showed a polyclonal response.
Deletions and extensive somatic mutations within the FrIII region may give false negative results with primers homologous to the region. A PCR using the method described, with a repertoire of primers homologous to the FrII and FrIII regions, will therefore increase the frequency of detection of monoclonality.
开发一种基于聚合酶链反应（PCR）的简单、优化方法，用于检测重排的免疫球蛋白重链（IgH）。
使用与框架II区和连接（JH）区保守序列同源的寡核苷酸（Fr2A、Fr2B）作为引物，研究了25例先前经Southern印迹法鉴定的B细胞淋巴增殖性疾病患者以及3例轻链骨髓瘤患者。
多克隆B细胞群体的PCR产物在3%琼脂糖凝胶上分析时显示出一条宽带；B细胞系和B淋巴增殖性疾病的DNA显示出一条离散带。通过对扩增产物进行克隆和测序以及用与框架3区同源的内部探针进行Southern印迹法，证实了扩增的特异性。引物Fr2A和Fr2B在3例轻链骨髓瘤患者中检测到单克隆性，而针对FrIII区的引物显示出多克隆反应。
FrIII区内的缺失和广泛的体细胞突变可能导致与该区域同源的引物产生假阴性结果。因此，使用所述方法并结合与FrII和FrIII区同源的引物库进行PCR，将增加单克隆性的检测频率。
