Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection
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Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection : implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia. / zur Stadt, Udo; Eckert, Conny; Rischewski, Johannes; Michael, Katharina; Golta, Steffi; Müller, Manuela; Schneppenheim, Reinhard; Kabisch, Hartmut.
in: J CHROMATOGR B, Jahrgang 792, Nr. 2, 25.07.2003, S. 287-98.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection
T2 - implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia
AU - zur Stadt, Udo
AU - Eckert, Conny
AU - Rischewski, Johannes
AU - Michael, Katharina
AU - Golta, Steffi
AU - Müller, Manuela
AU - Schneppenheim, Reinhard
AU - Kabisch, Hartmut
PY - 2003/7/25
Y1 - 2003/7/25
N2 - Incomplete T-cell-receptor delta (TCR-delta) rearrangements are widely used for detection of minimal residual disease in childhood acute lymphoblastic leukemia. In a substantial number of cases both alleles are rearranged and polymerase chain reaction (PCR) products have either to be cloned or excised and reamplified from acrylamide gels. Here we describe a novel HPLC-based method for identification and characterization of clonal TCR-delta targets. Clonality of PCR amplified TCR-delta products was examined on a high-resolution micropellicular DNASep matrix (WAVE Nucleic Acid Fragment Analysis System, Transgenomic) and subsequently classified into clonal, biclonal or negative samples. Vdelta2-Ddelta3 and Ddelta2-Ddelta3 rearrangements were analyzed by denaturing high-performance liquid chromatography (DHPLC), using triethylammonium acetate as an ion-pairing reagent, with a linear acetonitrile gradient at 50 degrees C. Biclonal elution profiles consisted of two individual homoduplex peaks and one heteroduplex peak unique for each patient sample. For characterization of biclonal rearrangements DHPLC separated samples were subjected to a second run and individual clonal peaks were collected. A software-controlled fragment collector was arranged in tandem with the HPLC system for this purpose and purified PCR products were collected in a time-dependent manner. Fractions were air dried and subsequently sequenced directly. The specificity of the observed patient specific sequences was tested via real time quantitative PCR on a LightCycler system.
AB - Incomplete T-cell-receptor delta (TCR-delta) rearrangements are widely used for detection of minimal residual disease in childhood acute lymphoblastic leukemia. In a substantial number of cases both alleles are rearranged and polymerase chain reaction (PCR) products have either to be cloned or excised and reamplified from acrylamide gels. Here we describe a novel HPLC-based method for identification and characterization of clonal TCR-delta targets. Clonality of PCR amplified TCR-delta products was examined on a high-resolution micropellicular DNASep matrix (WAVE Nucleic Acid Fragment Analysis System, Transgenomic) and subsequently classified into clonal, biclonal or negative samples. Vdelta2-Ddelta3 and Ddelta2-Ddelta3 rearrangements were analyzed by denaturing high-performance liquid chromatography (DHPLC), using triethylammonium acetate as an ion-pairing reagent, with a linear acetonitrile gradient at 50 degrees C. Biclonal elution profiles consisted of two individual homoduplex peaks and one heteroduplex peak unique for each patient sample. For characterization of biclonal rearrangements DHPLC separated samples were subjected to a second run and individual clonal peaks were collected. A software-controlled fragment collector was arranged in tandem with the HPLC system for this purpose and purified PCR products were collected in a time-dependent manner. Fractions were air dried and subsequently sequenced directly. The specificity of the observed patient specific sequences was tested via real time quantitative PCR on a LightCycler system.
KW - Base Sequence
KW - Child
KW - Chromatography, High Pressure Liquid
KW - DNA Probes
KW - Gene Rearrangement, delta-Chain T-Cell Antigen Receptor
KW - Humans
KW - Monitoring, Physiologic
KW - Neoplasm, Residual
KW - Polymerase Chain Reaction
KW - Precursor Cell Lymphoblastic Leukemia-Lymphoma
KW - Protein Denaturation
M3 - SCORING: Journal article
C2 - 12860036
VL - 792
SP - 287
EP - 298
JO - J CHROMATOGR B
JF - J CHROMATOGR B
SN - 1570-0232
IS - 2
ER -