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Amplification by the polymerase chain reaction of hypervariable regions of
the human genome for evaluation of chimerism after bone marrow
transplantation
L Ugozzoli, P Yam, LD Petz, GB Ferrara, RE Champlin, SJ Forman, D Koyal and RB Wallace
Department of Molecular Biochemistry, City of Hope National Medical Center,
Duarte, CA.
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.
Volume 77,
Issue 7,
pp. 1607-1615,
04/01/1991
Copyright © 1991 by The American Society of Hematology
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