Polymerase chain reaction versus Southern blot hybridization. Detection of immunoglobulin heavy-chain gene rearrangements.
Polymerase chain reaction versus Southern blot hybridization. Detection of immunoglobulin heavy-chain gene rearrangements.
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聚合酶链反应与 Southern 印迹杂交。
DOI：
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
J. Cossman
中科院分区：
文献类型：
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作者：
N. Sioutos;A. Bagg;G. Michaud;S. Irving;D. Hartmann;Hamid Siragy;D. R. Oliveri;J. Locker;J. Cossman
为了有效地确定b细胞淋巴细胞增殖性疾病的克隆性，我们改进了免疫球蛋白重链（IGH）基因重排聚合酶链反应（PCR）试验，该试验只需要单个引物种系变量（VH）和连接（JH）对，不涉及巢式引物，印迹杂交，放射性或扩增的PCR产物测序。这种简单的PCR技术可以在10 pg（相当于一个细胞）的DNA中检测到IGH基因重排，或者当克隆与多克隆b细胞的比例实验设定为1:100时。我们在83.5%（85个中的71个）克隆b细胞过程中检测到IGH基因重排，灵敏度接近于更繁琐的多重引物和嵌套引物检测。此外，该技术对固定组织（B5或福尔马林）同样有效，可用于微小样本、组织学切片、细针抽吸或脑脊液。与使用基因组JH探针的传统Southern blot分析相比，PCR分析显示，82%（45个中的37个）的b细胞过程中IGH基因重排被Southern blot阳性。29例阴性对照组织未见假阳性结果。我们现在在实验室常规使用IGH基因PCR来检测几乎任何组织样本中的克隆b细胞，作为早期诊断、分期和监测的辅助，而Southern blot程序仅用于少数诊断病例。只对少数诊断病例有效。
To determine efficiently the clonality of B-cell lymphoproliferative disorders, we modified an immunoglobulin heavy-chain (IGH) gene rearrangement polymerase chain reaction (PCR) assay that requires only a single primer germline variable (VH) and joining (JH) pair and does not involve nested priming, blot hybridization, radioactivity, or sequencing of the amplified PCR product. This simple PCR technique enabled detection of IGH gene rearrangements in as little as 10 pg (one cell equivalent) of DNA or when the clonal-to-polyclonal B-cell ratio was experimentally set at 1:1000. We detected IGH gene rearrangements in 83.5% (71 of 85) of clonal B-cell processes, a sensitivity approaching that of more cumbersome multiple primer and nested primer assays. Moreover, this technique is equally effective with fixed tissues, either B5 or formalin, and can be performed on minute samples, histologic sections, fine-needle aspirates, or cerebrospinal fluids. When compared with conventional Southern blot analysis using a genomic JH probe, the PCR assay demonstrated IGH gene rearrangements in 82% (37 of 45) of B-cell processes positive by Southern blot. No false-positive results were observed in 29 negative control tissues. We now use IGH gene PCR routinely in our laboratory for the detection of clonal B-cells in virtually any tissue sample as an aid in early diagnosis, staging, and monitoring, and the Southern blot procedure is reserved for only a minority of diagnostic cases. for only a minority of diagnostic cases.