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Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia

Standard

Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia. / zur Stadt, U; Rischewski, J; Schneppenheim, R; Kabisch, H.

in: CLIN CHEM, Jahrgang 47, Nr. 11, 01.11.2001, S. 2003-11.

Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung

Harvard

zur Stadt, U, Rischewski, J, Schneppenheim, R & Kabisch, H 2001, 'Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia', CLIN CHEM, Jg. 47, Nr. 11, S. 2003-11.

APA

zur Stadt, U., Rischewski, J., Schneppenheim, R., & Kabisch, H. (2001). Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia. CLIN CHEM, 47(11), 2003-11.

Vancouver

zur Stadt U, Rischewski J, Schneppenheim R, Kabisch H. Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia. CLIN CHEM. 2001 Nov 1;47(11):2003-11.

Bibtex

@article{112db35c49c1488db4a695a945c869ff,
title = "Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia",
abstract = "BACKGROUND: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.METHODS: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C.RESULTS: For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.CONCLUSIONS: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.",
keywords = "Base Sequence, Chromatography, High Pressure Liquid, Heteroduplex Analysis, Humans, Molecular Sequence Data, Polymerase Chain Reaction, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Receptors, Antigen, T-Cell, gamma-delta",
author = "{zur Stadt}, U and J Rischewski and R Schneppenheim and H Kabisch",
year = "2001",
month = nov,
day = "1",
language = "English",
volume = "47",
pages = "2003--11",
journal = "CLIN CHEM",
issn = "0009-9147",
publisher = "American Association for Clinical Chemistry Inc.",
number = "11",

}

RIS

TY - JOUR

T1 - Denaturing HPLC for identification of clonal T-cell receptor gamma rearrangements in newly diagnosed acute lymphoblastic leukemia

AU - zur Stadt, U

AU - Rischewski, J

AU - Schneppenheim, R

AU - Kabisch, H

PY - 2001/11/1

Y1 - 2001/11/1

N2 - BACKGROUND: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.METHODS: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C.RESULTS: For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.CONCLUSIONS: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.

AB - BACKGROUND: Denaturing HPLC (DHPLC) can be used to screen DNA for known and unknown mutations. We describe a novel, HPLC-based method for discrimination among polyclonal, oligoclonal, and/or clonal T-cell receptor gamma (TCR-gamma) rearrangements in samples from children with newly diagnosed acute lymphoblastic leukemia.METHODS: TCR rearrangements were PCR amplified from initial leukemic samples and, after heteroduplex-induction, the clonality status of each product was evaluated. To attain this, we used DHPLC on a high-resolution micropellicular matrix. Running conditions were established by melting-curve analysis of known clonal and polyclonal products and melting-point prediction software. Elution profiles were studied at 50 degrees C (native) and, to achieve optimal separation, at different column temperatures between 56 and 64 degrees C.RESULTS: For VgammaI-Jgamma1.3/2.3 rearrangements, an analysis temperature of 60 degrees C with a linear triethylammoniumacetate-acetonitrile gradient separated clonal bands from the polyclonal background amplification. Less than 15% clonal PCR product was detectable in mixtures of initial leukemic cell DNA and polyclonal DNA. Biallelic rearrangements produced two sharp peaks. Clonality of PCR products from 100 initial leukemic samples was completely identified in all investigated cases.CONCLUSIONS: Heteroduplex analysis with standardized DHPLC conditions simplifies the detection of unknown clonal or polyclonal TCR rearrangements in newly diagnosed leukemias. Clonal targets for detection of minimal residual disease are available after a short, automated analysis of PCR amplified rearrangements.

KW - Base Sequence

KW - Chromatography, High Pressure Liquid

KW - Heteroduplex Analysis

KW - Humans

KW - Molecular Sequence Data

KW - Polymerase Chain Reaction

KW - Precursor Cell Lymphoblastic Leukemia-Lymphoma

KW - Receptors, Antigen, T-Cell, gamma-delta

M3 - SCORING: Journal article

C2 - 11673369

VL - 47

SP - 2003

EP - 2011

JO - CLIN CHEM

JF - CLIN CHEM

SN - 0009-9147

IS - 11

ER -