Detection of clonal T-cell receptor-gamma gene rearrangement by PCR/temporal temperature gradient gel electrophoresis (TTGE)

Abstract

The present invention provides methods and compositions for detecting and analyzing clonal T-cell receptor (TCR) gene rearrangement using temporal temperature gradient gel electrophoresis (TTGE), which employs a gradual and uniform increase in the temperature of a constant denaturing gel to resolve different DNA molecules based on base pair composition. The present invention also provides methods and compositions for providing appropriate DNA migration markers for TTGE analysis.

What is claimed is:  
     
         1 . A method of preparing a DNA migration marker for use in a TTGE gel, comprising: 
 providing one or more TCR DNA segments having known migration rates in said TTGE gel.    
     
     
         2 . The method of  claim 1 , wherein said providing step comprises: 
 (a) amplifying one or more DNA samples comprising clonal TCR rearrangements using TCR-specific primers to provide one or more clonal DNA fragments;    (b) inserting each said clonal DNA fragment(s) in one or more plasmids;    (c) isolating each said plasmid(s); and    (d) amplifying the clonal DNA fragment in each said plasmid(s) to provide said one or more TCR DNA segments.    
     
     
         3 . A TTGE DNA migration marker, comprising: 
 a substantially pure DNA molecule comprising a TCR gene sequence; and    a buffer suitable for loading on a TTGE gel.