Detection of rearrangement of immunoglobulin heavy chain and T-cell receptor beta chain in leukemic cells by restricted polymerase chain reaction.
Rearrangement of the immunoglobulin heavy chain and of the T-cell receptor beta subunit was analyzed by using restricted polymerase chain reaction (PCR). To differentiate between the germline configuration and the rearranged genome in a DNA sample extracted from lymphocytes, we compared the ratio of the amplified products. The intensity of amplification of the intron region (JHF) upstream of the first joining region was compared to the intensity of joining region 6 of the immunoglobulin heavy chain. The number of the amplification cycles in the PCR was designated in such a way that the ratio of JHF/JH6 was less than one in the rearranged configuration. As the concentration of clonal B-lymphocytes with the rearranged genome in the sample increased the amplification of the JHF intron proportionally decreased. We used the same approach for the two constant regions of the T-cell receptor beta chain. As one of the intron regions of the constant sequence became depleted by rearrangement so the amplification of the particular region decreased. Therefore, the absence or decreased concentration of a particular product of amplification indicated deletion and thus rearrangement of the genome in the leukemic B- or T-lymphocytes. The threshold of detection of cells with the rearranged genome on a photograph of agarose gel loaded with the particular amplified regions and staining with the ethidium bromide is less than 10% by densitometric tracing and 25-50% by visual evaluation. This novel approach allows the detection of the rearranged DNA sequences in a 2 day span. Hence, it can serve as a diagnostic tool for the identification of clonal expansion of lymphocytes in acute leukemias and lymphomas in particular and for the detection of deleted genomic regions in general.
Stolc
V
V
Department of Pathology, School of Medicine, University of Pittsburg, PA 15261.
Uhrmacher
J
J
Krause
J R
JR
D000595:Amino Acid Sequence; D015321:Gene Rearrangement; D015329:Gene Rearrangement, T-Lymphocyte; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007133:Immunoglobulin Joining Region; D007938:Leukemia / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000235:genetics
D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D011948:Receptors, Antigen, T-Cell