Department of Haematology and Genetic Pathology, Flinders University and Medical Centre, Bedford Park, South Australia, Australia.
Br J Haematol. 2010 Apr;149(2):231-6. doi: 10.1111/j.1365-2141.2009.08071.x. Epub 2010 Jan 11.
Isolation and sequencing of the translocation breakpoint in chronic myeloid leukaemia-(CML) and acute promyelocytic leukaemia (APML) may help to elucidate the mechanism of translocation and provide a molecular marker for monitoring of minimal residual disease. Amplification across the translocation breakpoint was performed in samples from 91 patients with CML and 15 patients with APML using single-tube multiplex polymerase chain reaction (PCR) involving 308 primers for CML and 40 primers for APML. Nonspecific amplification was removed by a modification of PCR, termed sequential hybrid primer PCR (SHP-PCR), which involved two sequential rounds of PCR, each of which used a low concentration of a specially designed hybrid primer. The resultant amplified material was sequenced. The method as finally developed was simple quick and robust. The translocation breakpoint was successfully isolated and sequenced in all 106 samples. The strategy of highly multiplexed PCR followed by SHP-PCR is thus an effective method for isolating the translocation breakpoint in CML and APML. It may also be applicable to other haematological disorders characterised by translocation, deletion or inversion.
慢性髓细胞白血病（CML）和急性早幼粒细胞白血病（APML）中转录易位断点的分离和测序有助于阐明易位的机制，并为监测微小残留病提供分子标记。使用涉及 308 个 CML 引物和 40 个 APML 引物的单管多重聚合酶链反应（PCR），对 91 例 CML 患者和 15 例 APML 患者的样本进行了跨易位断点的扩增。通过一种称为顺序杂交引物 PCR（SHP-PCR）的 PCR 修饰去除非特异性扩增，该修饰涉及两轮 PCR，每轮都使用低浓度专门设计的杂交引物。对扩增产物进行测序。最终开发的方法简单、快速且稳健。在所有 106 个样本中都成功分离和测序了易位断点。因此，高度多重 PCR 后进行 SHP-PCR 的策略是分离 CML 和 APML 中转录易位断点的有效方法。它也可能适用于其他以易位、缺失或倒位为特征的血液疾病。
