Nucleotide sequence determination employing matched dideoxynucleotide terminator concentrations
First Claim
1. An apparatus for performing capillary electrophoresis of two samples comprising:
- a first separation capillary column containing a first sample to be separated into its components;
a second separation capillary column containing a second sample to be separated into its components;
means for causing separation by electrophoresis of the first and second samples simultaneously;
radiation means for irradiating detection points along the first and second capillary columns; and
detection means for individually detecting radiation corresponding to separated first sample in the first capillary column and separated second sample in the second capillary column.
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Abstract
A method of positively deciphering a DNA sequence of a sample by comparing data of two electropherograms obtained from separate electrophoresis of products from two complete sequence reactions of the same sample, whereby dideoxynucleotide terminators of different but matched concentration ratios are employed for the two reactions. The sequencing chemistry is chosen to result in terminator-concentration dependent in peak intensity in the electropherograms. By appropriately choosing the matched concentration ratios of the terminators used in the two sequence reactions, ambiguities in the discrimination of the peaks in the result of one sequence reaction can be resolved by comparing the result of the other sequence reaction. The sequence of the sample can therefore be deciphered to a high degree of accuracy. For fluorescence detection of electrophoresis of the sequence reactions, three ddNTP terminators and one fluor are included in the sequencing chemistry of each reaction. The position of the fourth terminator in the sequence is determined from the location of the gaps or tiny peaks in the background signal in the electropherogram which appear between the peaks produced by the three ddNTP terminators used. A dual channel electrophoresis device facilitates obtaining two matched electropherograms of two simultaneous sequence reactions which employ matched concentration ratios of ddNTP terminators. The device comprises a single radiation source and two parallel electrophoresis channels simultaneously operating under substantially similar conditions.
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Citations
16 Claims
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1. An apparatus for performing capillary electrophoresis of two samples comprising:
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a first separation capillary column containing a first sample to be separated into its components; a second separation capillary column containing a second sample to be separated into its components; means for causing separation by electrophoresis of the first and second samples simultaneously; radiation means for irradiating detection points along the first and second capillary columns; and detection means for individually detecting radiation corresponding to separated first sample in the first capillary column and separated second sample in the second capillary column. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
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10. An apparatus for performing capillary electrophoresis of two samples comprising:
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a first separation capillary column containing a first sample to be separated; a second separation capillary column containing a second sample to be separated; means for causing separation by electrophoresis of the first and second sample simultaneously; and a same radiation source for detection operatively coupled to both the first and second capillary columns.
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11. An apparatus for determining DNA sequence of a sample DNA by performing capillary electrophoresis on first and second sequence mixtures which are obtained by performing first and second sequencing reactions on the sample DNA, each using at least three ddNTPs from a group of four ddNTPs W, X, Y, and Z;
- wherein for the first sequencing reaction, three ddNTPs X, Y and Z are selected from the group of four ddNTPs, the first sequencing reaction producing a first sequence mixture;
wherein for the second sequence reaction, three ddNTPs W, Y and Z are selected, the second sequencing reaction producing a second sequence mixture;
the apparatus comprising;a first separation capillary column containing the first sequence mixture to be separated into its components; a second separation capillary column containing the second sequence mixture to be separated into its components; means for causing separation by electrophoresis of the first and second sequence mixtures simultaneously; radiation means for irradiating first and second detection points along the first and second capillary columns, respectively; detection means for individually detecting radiation corresponding to separated first sequence mixture in the first capillary column and separated second sequence mixture in the second capillary column; means for obtaining a first set of data peaks each representing the presence of ddNTP terminated oligonucleotides in the first sequence mixture of a particular size passing through the first detection point in the first column, the relative sizes of the data peaks being dependent on the concentration of the respective ddNTP; means for obtaining a second set of data peaks representing the presence of ddNTP terminated oligonucleotides of a particular size in the second sequence mixture passing through the second detection point in the second capillary column, the relative sizes of the data peaks being dependent on the concentration of the respective ddNTP. - View Dependent Claims (12, 13, 14, 15, 16)
- wherein for the first sequencing reaction, three ddNTPs X, Y and Z are selected from the group of four ddNTPs, the first sequencing reaction producing a first sequence mixture;
Specification