Method and apparatus for sequencing of dna using an internal calibrant

Abstract

For evaluation of a target DNA sequence, a sample mixture is prepared containing one or more sets of sequencing polynucleotide fragments, each set containing fragments having lengths indicative of the positions of at least one base within the target DNA sequence. These sequencing fragment sets are each labeled with a different type of label (for example fluorescent labels). The sample mixture also includes a set of calibrant polynucleotide fragments having a plurality of known fragment lengths. The calibrant polynucleotide fragments are labeled with a spectroscopically-distinguishable calibrant label. The sample mixture is then electrophoretically separated to separate the polynucleotide fragments as a function of fragment length. Real-time detection is used to detect the label(s) on the set(s) of sequencing fragments and the calibrant label as they migrate in a common lane of the separation medium to produce a sequencing data trace and a calibrant data trace. The calibrant peaks are then used to define a set of coefficients for linearizing the sequencing data trace from each lane to a common corrected time scale in which the peaks from each lane are evenly spaced. The linearized sequencing data traces are then aligned by assigning base position numbers to each peak in the sequencing data traces, and these aligned traces are used for base calling.