QUANTITATION OF IMMUNOGLOBULIN MU-GAMMA-1 HEAVY-CHAIN SWITCH REGION RECOMBINATION BY A DIGESTION CIRCULARIZATION POLYMERASE CHAIN-REACTION METHOD
QUANTITATION OF IMMUNOGLOBULIN MU-GAMMA-1 HEAVY-CHAIN SWITCH REGION RECOMBINATION BY A DIGESTION CIRCULARIZATION POLYMERASE CHAIN-REACTION METHOD
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DOI：
10.1073/pnas.89.15.6978
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发表时间：
1992-08-01
影响因子：
11.1
通讯作者：
MAX, EE
中科院分区：
文献类型：
--
作者：
CHU, CC;PAUL, WE;MAX, EE
在免疫应答过程中，无论有无IgD，表达表面IgM的B淋巴细胞均可转为表达其他亚型(Ig G、Ig A或Ig E)。对克隆的骨髓瘤和杂交瘤基因组DNA的分析表明，同型转换伴随着一种重排，其特征是Mu-基因的转换区(S)和与新的同型相关的转换区之间的DNA缺失，从而形成一个复合的S区。在正常B细胞群体中测量这种缺失重排一直是困难的，但对于研究重排的机制和确定缺失重排是否是所有类别转换的原因将是有用的。我们建立了一种检测缺失重排的灵敏方法，我们称之为消化-环化聚合酶链式反应(PCR)。在这个实验中，基因组dna被一种限制酶消化，该限制酶识别重组的复合S区域两侧的位点。然后，将消化后的DNA在低浓度下连接，以利于形成环状。该连接连接了每个限制性片段的5‘和3’端，使得使用适当的引物通过连接的限制性内切酶位点进行PCR扩增成为可能。从经过缺失重排的DNA中产生单一大小的聚合酶链式反应产物并可以进行定量。在此，我们证明了消化-环化聚合酶链式反应方法可以检测在脂多糖和白介素4作用下的B细胞中S-穆-S-γ-1重排。该方法对仅占总数1-2%的开关细胞的定量是足够灵敏的。
B lymphocytes expressing surface IgM with or without IgD may switch to the expression of other isotypes (IgG, IgA, or IgE) in the course of immune responses. Analyses of genomic DNA from cloned myelomas and hybridomas have shown that the isotype switch is accompanied by a rearrangement characterized by deletion of DNA between the switch (S) region of the mu-gene and that associated with the new isotype, resulting in the formation of a composite S region. Measurement of this deletional rearrangement has been difficult in populations of normal B cells but would be useful for investigating the mechanism of the rearrangement and determining whether deletional rearrangement is responsible for all instances of class switching. We have developed a sensitive assay for deletional rearrangement that we designate the digestion-circularization polymerase chain reaction (PCR). In this assay, genomic DNA is digested with a restriction enzyme that recognizes sites that flank the recombined composite S region. The digested DNA is then ligated at low concentrations to favor the formation of circles. The ligation joins the 5' and 3' ends of each restriction fragment, making it possible to amplify by PCR across the ligated restriction site by using appropriate primers. From DNA that has undergone deletional rearrangement, a single-sized PCR product is produced and can be quantitated. We demonstrate here that the digestion-circularization PCR assay can detect S-mu-S-gamma-1 rearrangements in B cells cultured with lipopolysaccharide and interleukin 4. The assay is sensitive enough to quantitate switched cells constituting only 1-2% of the population.