Inverse PCR amplification of rare T cell receptor delta messages from mucosal biopsy specimens.
Reverse transcription (RT) PCR is an important tool in studies of the repertoire of T cell antigen receptors (TCR). In combination with inverse PCR, it can be used to amplify TCR transcripts whose V regions are unknown. Little is currently known regarding the delta TCR repertoire in human intestine. Here, RT inverse PCR was adapted to efficiently amplify low abundance delta TCR sequences from human colonic mucosal biopsy specimens. In this protocol, high yields of PCR products were obtained by optimizing the conditions at which cDNA autoligation was performed.
Chowers
Y
Y
Department of Medicine 0623D, University of California, San Diego, La Jolla 92093-0623.
Holtmeier
W
W
Morzycka-Wroblewska
E
E
Kagnoff
M F
MF
D001483:Base Sequence; D003106:Colon; D011088:DNA Ligases / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism*
D017931:DNA Primers; D012333:RNA, Messenger; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011088:DNA Ligases