Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation.
Amplification by the polymerase chain reaction of hypervariable regions of the human genome for evaluation of chimerism after bone marrow transplantation.
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DOI：
10.1182/blood.v77.7.1607.1607
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发表时间：
1991
期刊：
影响因子：
20.3
通讯作者：
L. Ugozzoli;P. Yam;LD Petz;GB Ferrara;RE Champlin;SJ Forman;D. Koyal;RB Wallace
中科院分区：
文献类型：
--
作者：
L. Ugozzoli;P. Yam;LD Petz;GB Ferrara;RE Champlin;SJ Forman;D. Koyal;RB Wallace
我们结合聚合酶链反应（PCR）和寡核苷酸杂交作为一种新的和敏感的技术来评估移植后嵌合体。用于杂交的特异性寡核苷酸被合成为与具有可变数目的串联重复序列（VNTR）的区域的串联重复核心序列同源。这些基因座的多态性是由重复数量的等位基因差异引起的。将位于每个相应VNTR的重复区侧翼的引物用于扩增。扩增供体和供体移植前DNA和受体移植后DNA。在凝胶电泳后通过凝胶内杂交或在Southern转移后分析所得片段。为了证实我们的发现，我们还进行了限制性片段长度多态性（RFLP）的标准测定。13例骨髓移植后混合嵌合体4例，完全嵌合体5例，白血病复发2例，内源性造血再生2例。通过混合不同比例的受体和供体DNA来确定该方法的灵敏度;混合物中次要组分的检测限为0.1%。PCR数据与RFLP数据在所有情况下，除了两个PCR证明比RFLP更敏感。VNTRs的PCR扩增结合寡核苷酸杂交是一种记录移植后嵌合体的新技术，与RFLP分析相比具有以下优点：灵敏度高，使用少量DNA（250 ng），易于制备DNA，消除了对限制性内切酶的需要，以及在2天内完成研究的能力。
We combined the polymerase chain reaction (PCR) with oligonucleotide hybridization as a novel and sensitive technique to evaluate posttransplant chimerism. Specific oligonucleotides for hybridization were synthesized homologous to tandemly repetitive core sequences of regions with a variable number of tandem repeats (VNTRs). Polymorphisms at such loci result from allelic differences in the number of repeats. Primers flanking the repeat region of each of the corresponding VNTRs were used for amplification. Recipient and donor pretransplant DNA and recipient posttransplant DNA were amplified. The resultant fragments were analyzed after gel electrophoresis either by hybridization in-gel or after Southern transfer. To confirm our findings, we also performed standard assays of restriction fragment length polymorphisms (RFLPs). Evaluation of 13 selected cases indicated mixed chimerism (4), complete chimerism (5), recurrence of leukemia (2), and endogenous repopulation of hematopoiesis (2) after marrow transplantation. Sensitivity of the method was determined by mixing various proportions of recipient and donor DNA; the limit of detection of the minor component in a mixture was 0.1%. PCR data correlated with RFLP data in all cases except two in which PCR proved more sensitive than RFLP. PCR amplification of VNTRs combined with oligonucleotide hybridization is a novel technique for documenting posttransplant chimerism and has advantages over RFLP analysis: high sensitivity, use of small amounts of DNA (250 ng), ease of preparation of DNA, elimination of need for restriction enzymes, and the ability to complete studies in 2 days.