DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.
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DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach. / Zur Stadt, Udo; Isbarn, Hendrik; Schneppenheim, Reinhard; Kabisch, Hartmut.
in: INT J ONCOL, Jahrgang 27, Nr. 2, 2, 08.2005, S. 547-552.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - DHPLC based fraction collection of TCR-gamma rearrangements in childhood ALL: direct sequencing of products amplified by a single or a multiplex PCR approach.
AU - Zur Stadt, Udo
AU - Isbarn, Hendrik
AU - Schneppenheim, Reinhard
AU - Kabisch, Hartmut
PY - 2005/8
Y1 - 2005/8
N2 - Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.
AB - Clonal T-cell receptor gamma (TCR-gamma) rearrangements are frequently used for detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukemia. In approximately 70-80% of cases PCR amplified clonal rearrangements can be sequenced directly. The remaining 20-30% are rearranged on both alleles for the same target and disables direct sequencing. Here we describe a novel HPLC based method for identification and characterisation of TCR-gamma rearrangements either by a single or a multiplex PCR approach. The latter one amplifies several Vgamma segments in two distinct reactions either with a Jgamma1.3/2.3 or a Jgamma1.1/2.1 specific primer. The clonality status was evaluated on a high resolution micropellicular DNASep matrix (WAVE, Transgenomic) at different temperatures. From 331 samples analysed, 151 samples were positive for VgammaI-Jgamma1.3/2.3 including 51 biclonal rearrangements. For characterisation of these biclonal products or for products generated by multiplex-PCR, a second HPLC run was performed utilising a tandem arranged fraction collector. From clearly separated biclonal/biallelic products, several collected fractions were air-dried and afterwards sequenced directly with the appropriate Jgamma primer. We conclude from our results that HPLC is a fast and reliable method for identification of TCR-gamma rearrangements. The fraction collection simplifies the characterisation of single alleles within biclonal or biallelic rearrangements or within multiplex PCR products. The target identification process prior to routine MRD analysis will be shortened due to a simplified screening and sequencing strategy.
KW - Base Sequence
KW - Child
KW - Chromatography, High Pressure Liquid
KW - DNA, Neoplasm
KW - Gene Rearrangement, delta-Chain T-Cell Antigen Receptor
KW - Humans
KW - Polymerase Chain Reaction
KW - Precursor Cell Lymphoblastic Leukemia-Lymphoma
KW - Sequence Analysis, DNA
U2 - 10.3892/ijo.27.2.547
DO - 10.3892/ijo.27.2.547
M3 - SCORING: Journal article
C2 - 16010438
VL - 27
SP - 547
EP - 552
JO - INT J ONCOL
JF - INT J ONCOL
SN - 1019-6439
IS - 2
M1 - 2
ER -