Methods for rapid identification and quantitation of nucleic acid variants
AssigneeIbis Biosciences, Inc.
InventorsDavid J. Ecker, Steven A. Hofstadler, Thomas A. Hall, Kristin Sannes Lowery
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Abstract
There is a need for nucleic acid analysis which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need, among others by providing a method of nucleic acid amplification of overlapping sub-segments of a nucleic acid followed by molecular mass measurement of resulting amplification products by mass spectrometry, and determination of the base compositions of the amplification products.
Claims34
A method for analyzing a nucleic acid comprising the steps of:
- (a) obtaining a sample comprising nucleic acid for base composition analysis;
- (b) selecting at least two primer pairs that will generate overlapping amplification products of at least two sub-segments of the nucleic acid;
- (c) amplifying at least two nucleic acid sequences of a region of the nucleic acid designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;
- (d) determining base compositions of the amplification products by;
- (i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer;
- and
- (ii) converting one or more of the measured molecular masses to base compositions;
- (e) comparing one or more of the base compositions with reference base composition data for the nucleic acid sequence;
- and
- (f) identifying the presence of a particular nucleic acid sequence or variant thereof.
A method for identifying a human comprising the steps of:
- (a) obtaining a sample comprising mitochondrial DNA of the human for base composition analysis;
- (b) selecting at least two primer pairs that will generate overlapping amplification products representing sub-segments of the mitochondrial DNA;
- (c) amplifying at least two nucleic acid sequences of a region of the mitochondrial DNA designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;
- (d) determining base compositions of the amplification products by;
- (i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer;
- and
- (ii) converting one or more of the measured molecular masses to base compositions;
- (e) comparing one or more of the base compositions with reference base composition data for the nucleic acid sequence thereby identifying the human.
A method for characterizing heteroplasmy in mitochondrial DNA comprising the steps of:
- (a) obtaining a sample comprising mitochondrial DNA for base composition analysis;
- (b) selecting at least two primer pairs that will generate overlapping amplification products representing sub-segments of the mitochondrial DNA;
- (c) amplifying at least two nucleic acid sequences of a region of the mitochondrial DNA designated as a target for base composition analysis using the at least two primer pairs, thereby generating at least two overlapping amplification products;
- (d) determining base compositions of the amplification products by;
- (i) measuring molecular masses of one or more of the amplification products generated in step (c) using a mass spectrometer;
- and
- (ii) converting one or more of the measured molecular masses to base compositions;
- (e) comparing one or more of the base compositions with one or more base compositions of reference sub-segments of a reference sequence;
- and
- (f) identifying at least two distinct amplification products with distinct base compositions obtained by the same pair of primers, thereby characterizing the heteroplasmy.
Dependencies are inferred from the claim text.