In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction.

A method is described using the polymerase chain reaction (PCR) to amplify defined nucleic acid strands in individual cells in situ in conventional smears of bone marrow and peripheral cells. Using radioactively labeled precursors, the incorporation into newly synthesized strands by PCR can be detected by microautoradiography. The specificity of the method can be monitored by gel electrophoresis of the material shed into the reaction mixture. Thus it could be shown that even single genes in individual cells can be amplified to visibility. In a mixture of HIV infected and non infected cells both can be clearly distinguished from one another.

A reliable approach for sequencing clone-specific CDRIII regions in B-cell lymphoma.

We report a reliable approach for sequencing lymphoma-specific CDRIII regions. CDRIII regions present in DNA prepared from routinely fixed and paraffin-embedded diagnostic lymph node material were amplified by the use of consensus VH and JH primers via PCR. PCR products were subcloned directly, without purification or modification of PCR fragments. Only small amounts of miniprep plasmid DNA of recombinant clones were required for cycle sequencing, resulting in autoradiograms of high quality. The easy and reproducible method which we describe has enabled us to determine the lymphoma-specific CDRIII region in 7/11 high-grade non-Hodgkin's lymphomas as well as in 3/3 cases of ALL and 1/1 case of a centroblastic/centrocytic lymphoma. The obtained sequence data can serve to generate lymphoma-specific oligonucleotides, which then can be used as PCR primers or hybridization probes for the detection of minimal residual disease in individual patients.