Title
Denaturing and non-denaturing gel electrophoresis as methods for the detection ofjunctional diversity in rearranged T cell receptor sequences
Author
Offermans, M.T.C.
Sonneveld, R.D.
Bakker, E.
Deutz-Terlouw, P.P.
de Geus, B.
Rozing, J.
TNO Preventie en Gezondheid
Publication year
1995
Abstract
Two nucleic acid gel electrophoresis techniques were tested as a possible tool for analyzing junctional diversity in rearranged T cell receptor (TcR) sequences in order to define the extent of T cell heterogeneity. For this purpose denaturing gradient gel electrophoresis (DGGE) as well as non-denaturing gel electrophoresis (nDGE) techniques have been studied. Detection of junctional diversity is based on mobility shifts, caused by nucleotide sequence polymorphism, of polymerase chain reaction amplified rearranged TcR sequences. DGGE as well as nDGE procedures are suitable for the detection of junctional diversity in TcR V gene family sequences based on sequence dependent separation. Compared to DGGE, nDGE of DNA is a relatively simple and rapid procedure, with a high separation potential. nDGE permits separation of double stranded (homoduplexes) and/or single stranded DNA molecules of the majority of TcR chain encoding sequences. Formation and detection of unique heteroduplex molecules combined with single stranded DNA molecule analysis in nDGE permits the recognition of the remaining sequences, thus providing additional information on the degree of T cell heterogeneity. In conclusion, these nucleic acid gel electrophoresis techniques allow a direct assessment of the heterogeneity and clonality of T cell populations by the detection of junctional diversity in TcR chain encoding sequences. This analysis can be performed without the need of cell propagation and/or cellular cloning procedures, thereby eliminating the risk of introducing technical artefacts.
Subject
Biology
Heteroduplex DNA molecule
Junctional diversity
Nucleic acid gel electrophoresis
T cell heterogeneity
T cell receptor
Animal
Base Sequence
Comparative Study
DNA
Electrophoresis, Polyacrylamide Gel
Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor
Intestinal Mucosa
Male
Mice
Mice, Inbred BALB C
Molecular Sequence Data
Polymorphism (Genetics)
Receptors, Antigen, T-Cell, gamma-delta
RNA
Sensitivity and Specificity
T-Lymphocytes
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http://resolver.tudelft.nl/uuid:b0682afc-238e-477d-8ed7-7917a405a086
DOI
https://doi.org/10.1016/0022-1759(94)00334-s
TNO identifier
232928
ISSN
0022-1759
Source
Journal of Immunological Methods, 181 (1), 101-114
Document type
article