Direct measurement of T-cell receptor repertoire diversity with AmpliCot
| Autor: | Joseph M. McCune, Paul D. Baum |
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| Rok vydání: | 2006 |
| Předmět: |
Time Factors
Receptors Antigen T-Cell alpha-beta Biology Sensitivity and Specificity Biochemistry Article Mice Structure-Activity Relationship chemistry.chemical_compound Nucleic acid thermodynamics Animals Molecular Biology Gene Fluorescent Dyes Gene Rearrangement Mice Knockout Genetics Cloning Reverse Transcriptase Polymerase Chain Reaction DNA–DNA hybridization T-cell receptor Nucleic acid sequence Nucleic Acid Hybridization DNA Cell Biology Gene rearrangement respiratory system Mice Inbred C57BL Kinetics Microscopy Fluorescence chemistry human activities Biotechnology |
| Zdroj: | Nature Methods. 3:895-901 |
| ISSN: | 1548-7105 1548-7091 |
| DOI: | 10.1038/nmeth949 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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