Method and apparatus for DNA sequencing

• US 20020192697A1
• Filed: 06/21/2002
• Published: 12/19/2002
• Est. Priority Date: 04/17/2000
• Status: Abandoned Application

• Current Assignee: Bayer Healthcare LLC (Bayer AG)
• Original Assignee: Bayer Healthcare LLC (Bayer AG)
• Inventors: Izmailov, Alexandre M.
• Application Number:
  US10/177,224
• Publication Number:
  US 20020192697A1
• Time in Patent Office: Days
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules

Abstract

The sequence of a target DNA molecule is determined by preparing four chain termination reaction mixtures, one for each base type. The first set of fragments, indicative of the positions of a first type of base, are labeled with a first fluorescent label. The second set of fragments, indicative of the positions of a second type of base, are labeled with a second fluorescent label different from the first fluorescent label, The third set of fragments, indicative of the positions of a third type of base, are labeled with a third fluorescent label, different from the first and second fluorescent labels or with at least two labels, including at least one fluorescent label selected from among the first, second and third fluorescent labels. The fourth set of fragments, indicative of the positions of a fourth type of base, are labeled differently from the third set of chain-termination fragments and with at least two different species of labels including at least one fluorescent label selected from among the first, second and third fluorescent labels. Thus, a total of only three fluorescent labels are required to label the DNA sequencing fragments. The first, second, third and fourth sets of chain-termination fragments are loaded onto the same lane of an electrophoresis separation medium and separated in an electric field. The separated fragments are detected in real-time as they migrate in the electrophoresis separation medium by irradiating the separated fragments with an excitation beam and collecting light emitted by the fluorescent labels in three optical channels. The signals from three optical channels are evaluated to determine a DNA sequence for the target species. This evaluation can be done with a specifically programmed computer.

Claims

1. A method for DNA sequencing comprising the steps of:

(a) preparing a first set of chain-termination fragments indicative of the positions of a first type of base, wherein the fragments in the first set of fragments are labeled with a first fluorescent label;

(b) preparing a second set of chain-termination fragments indicative of the positions of a second type of base, different from the first type of base, wherein the fragments in the second set of fragments are labeled with a second fluorescent label different from the first fluorescent label;

(c) preparing a third set of chain-termination fragments indicative of the positions of a third type of base, different from the first and second types of bases, wherein the fragments in the third set of fragments are labeled with a third fluorescent label, different from the first and second fluorescent labels or are double-labeled with at least two different species of fluorescent labels selected from among the first, second and third fluorescent labels;

(d) preparing a fourth set of chain-termination fragments indicative of the positions of a fourth type of base, different from the first, second and third types of bases, wherein the fragments in the fourth set of fragments are labeled differently from the third set of chain-termination fragments and are double-labeled with at least two different species of fluorescent labels selected from among the first, second and third fluorescent labels;

(e) loading the first, second, third and fourth sets of chain-termination fragments in the same lane of an electrophoresis separation medium;

(f) separated the loaded fragments on the electrophoresis separation medium;

(g) detecting the separated fragments as they migrate in the electrophoresis separation medium by irradiating the separated fragments with an excitation beam and collecting light emitted by the fluorescent labels in three optical channels, one for detecting each of the three fluorescent labels; and

(h) evaluating signals from the three optical channels to determine a DNA sequence.

2. The method according to claim 1, wherein the fluorescent labels are cyanine dyes.

3. The method of claim 1, wherein the fragments in the first and second set of chain termination fragments are labeled by a fluorescent label associated with an extended oligonucleotide primer.

4. The method of claim 1, wherein in preparing the fourth set of chain termination fragments, one of the fluorescent labels is associated with al extended oligonucleotide primer and the other of the fluorescent labels is associated with a chain-terminator.

5. The method of claim 1, wherein in preparing the fourth set of chain termination fragments, both of the fluorescent labels are associated with an extended oligonucleotide prime.

6. The method of claim 5, wherein the extended oligonucleotide primer is a double-labeled primer.

7. The method of claim 1, wherein in preparing the fourth set of chain termination fragments, both of the fluorescent labels are associated with a chain-terminator.

8. The method of claim 4, wherein the fragments of the third set of chain termination fragments arc labeled with the third fluorescent label.

9. The method according to claim 8, wherein the fluorescent labels are cyanine dyes.

10. The method of claim 4, wherein the fragments of the third set of chain termination fragments are labeled at least two different species of fluorescent labels selected from among the first, second and third fluorescent labels.

11. The method according to claim 10, wherein the fluorescent labels are cyanine dyes.

12. The method of claim 10, wherein in preparing at least one of the third or fourth sets of chain termination fragments, one of the fluorescent labels is associated with an extended oligonucleotide primer and the other of the fluorescent labels is associated with a chain-terminator.

13. The method of claim 10, wherein in preparing at least one of the third or fourth sets of chain termination fragments, both of the fluorescent labels are associated with an extended oligonucleotide primer.

14. The method of claim 13, wherein the extended oligonucleotide primer is a double-labeled primer.

15. The method of claim 10, wherein in preparing at least one of the third or fourth sets of chain termination fragments, both of the fluorescent labels are associated with a chain-terminator.

16. The method of claim 1, wherein the double-labeling of at least one of the third or fourth sets of fragments is virtual double labeling.

17. The method according to claim 1, wherein the double-labeling of at least one of the third or fourth sets of fragments is accomplished by affixing two labels to each fragment, and wherein the fragments in the first and second fragments sets, and the third fragment set if singly labeled, are modified by addition of an additional label to compensate of difference in electrophoretic mobility.

18. An apparatus for obtaining information for use in sequencing of DNA comprising:

(a) an electrophoresis apparatus comprising a separation medium for separation of sets of chain termination fragments in at least one lane; and

(b) an optical section comprising

at least one excitation source for providing excitation energy to fragments at a detection site in the lane in the separation medium;

a detector system having exactly three optical channels for the detection of emitted light of three different wavelengths.

19. An apparatus for sequencing DNA comprising:

(a) an electrophoresis apparatus comprising a separation medium for separation of sets of chain termination fragments in at least one lane;

(b) an optical section comprising

at least one excitation source for providing excitation energy to fragments at a detection site in the lane in the separation medium;

a detector system having three optical channels for the detection of emitted light of three different wavelengths; and

(c) a data processor operatively connected to receive three data streams from the three optical channels, and to process the three data streams into a DNA sequence.