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Polymerase Chain Reaction Versus Southern Blot Hybridization Detection of Immunoglobulin Heavy‐Chain Gene Rearrangements

 

作者: Nicholas Sioutos,   Adam Bagg,   Ginette Michaud,   Steven Irving,   Dan Hartmann,   Hamid Siragy,   Douglas Oliveri,   Joseph Locker,   Jeffrey Cossman,  

 

期刊: Diagnostic Molecular Pathology  (OVID Available online 1995)
卷期: Volume 4, issue 1  

页码: 8-13

 

ISSN:1052-9551

 

年代: 1995

 

出版商: OVID

 

关键词: Polymerase chain reaction;Southern blot hybridization;B-cell lymphoproliferative processes;Immunoglobulin heavy-chain gene rearrangement.

 

数据来源: OVID

 

摘要:

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 JHprobe, 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.

 

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