The use of anchored polymerase chain reaction for the study of large numbers of human T-cell receptor transcripts.
Anchored-PCR (A-PCR) is an approach designed to amplify and clone sequences with unknown 5' or 3' extremities. A-PCR is therefore appropriate for studying variable region of T-cell receptors (TCRs) expressed in polyclonal T-cell populations since it does not prejudge which variable gene segments are actually being used. We report here some critical modifications in the initial procedure to make it easy to clone and sequence large series of TCR transcripts. They have been introduced to improve both the yield and specificity of TCR amplified products and include re-amplification, size selections of the material combined with the successive use of nested TCR constant region specific primers. This procedure has been successfully applied to the study of the repertoire of both TCR alpha/beta+ and gamma/delta+ human T-cells. The efficiency of the present A-PCR protocol will help to precisely analyze TCR usage in normal and pathological situations.
Ferradini
L
L
Laboratoire d'Hémato-Immunologie, INSERM U333, Institut Gustave-Roussy, Villejuif, France.
Roman-Roman
S
S
Azogui
O
O
Genevée
C
C
Viel
S
S
Hercend
T
T
Triebel
F
F
D001483:Base Sequence; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics; D017422:Sequence Analysis, DNA; D013601:T-Lymphocytes / Q000502:physiology
D009692:Nucleic Acid Heteroduplexes; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta