Molecular analysis of polymorphic loci to study chimerism after allogeneic bone marrow transplantation. Heteroduplex analysis in denaturing gradient gel electrophoresis: a new approach to detecting residual host cells.
Since both donor/recipient origin of hematopoietic cells and the proportion of residual host cells influence the outcome of BMT, we performed this study to design a rapid and efficient strategy for chimerism analysis. Using the polymerase chain reaction, we analyzed the informativity and sensitivity of two kinds of polymorphism in 35 HLA-identical sibling pairs: four variable number of tandem repeats (VNTR) systems and the frameworks (FW) of the beta-globin gene. The latter polymorphisms were analyzed by means of denaturing gradient gel electrophoresis, a method that identifies heterozygosity by the presence of heteroduplexes. All the siblings were found to be informative for at least one polymorphic system. In addition, 34 of the 35 sibling pairs (97%) bore at least one VNTR or FW system which permitted the detection of the minor population (i.e., the first sibling's DNA contained an allele that was absent from the second sibling's DNA). The VNTR- and FW-based methods were highly sensitive, being able to reveal minor populations representing as little as 1% and 5% of total DNA, respectively. The value of the beta-globin gene FW analysis lies in the detection of additional heteroduplexes, which reflect genotypic differences between siblings' DNA. We describe the experimental conditions required to detect the minor DNA population in virtually all cases. As direct evidence of mixed chimerism can be obtained from denaturing gradient gel electrophoresis analysis of any highly informative polymorphic system, this method can be applied to chimerism analysis.
Muniz
E S
ES
Unité de Greffe de Moelle, Inserm U91, Hôpital Henri Mondor, Créteil, France.
Plassa
F
F
Amselem
S
S
Goossens
M
M
Vernant
J P
JP
D001483:Base Sequence; D016026:Bone Marrow Transplantation / Q000502:physiology*; D002499:Centrifugation, Density Gradient; D002678:Chimera / Q000235:genetics; D004587:Electrophoresis, Agar Gel / Q000379:methods; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000032:analysis; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid; D012680:Sensitivity and Specificity
D009692:Nucleic Acid Heteroduplexes; D005914:Globins