Direct measurement of T-cell receptor repertoire diversity with AmpliCot.
Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) beta chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.
Baum
Paul D
PD
Division of Experimental Medicine, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California 94110, USA.
McCune
Joseph M
JM
D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015321:Gene Rearrangement; D007700:Kinetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence / Q000379:methods*; D009693:Nucleic Acid Hybridization; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D013997:Time Factors
D005456:Fluorescent Dyes; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA