Biomarker anaylsis using scodaphoresis

摘要

The invention discloses methods and apparatus for characterizing trace nucleic acids that are biomarkers for disease. The methods and apparatus provide increased sensitivity to such trace nucleic acids, and allow analysis of nucleic acids present in a sample at only 0.01% of the wild-type sequences. The methods and apparatus are also designed for straightforward multiplexing, thus allowing pooling of clinical samples.

主权项

1. A method of characterizing a biomarker in a sample, the method comprising:
providing a sample comprising a wild-type nucleic acid and a corresponding mutant nucleic acid that is a biomarker for a disease; introducing a plurality of positive controls to the sample, the plurality of positive controls comprising a sequence identical to the mutant nucleic acid and a unique control sequence comprising a number of degenerate bases; amplifying the mutant nucleic acid and the plurality of positive controls in the sample; loading the amplified nucleic acid sample and the plurality of positive controls on a separation medium, the separation medium comprising an immobilized probe, the immobilized probe comprising a nucleic acid sequence complementary to the mutant nucleic acid; enriching the amplified nucleic acid sample for the mutant nucleic acid and the plurality of positive controls over the wild type nucleic acid by applying a time-varying driving field and a time-varying mobility-varying field to the separation medium; characterizing the enriched nucleic acid in the sample with a technique selected from nucleic acid sequencing, quantitative PCR (qPCR), mass spectrometry, and hybridization assay; and quantifying abundance of the mutant nucleic acid in the provided sample using the plurality of positive controls.