Evanescence-based multiplex sequencing method

• US 20070020626A1
• Filed: 12/18/2002
• Published: 01/25/2007
• Est. Priority Date: 12/19/2001
• Status: Active Grant

• Current Assignee: Rudolf Rigler
• Original Assignee: Rudolf Rigler
• Inventors: Rigler, Rudolf
• Granted Patent: US 8,017,319 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention relates to a method and a device for evanescence-based multiplex sequencing of nucleic acid molecules immobilized on a support.

Claims

1. Method for sequencing nucleic acids comprising the steps:

(a) providing an at least partially optically transparent support with a multitude nucleic acid molecules immobilized thereon where the nucleic acid molecules carry several fluorescent marker groups,

(b) progressive cleavage of individual nucleotide building blocks from the immobilized nucleic acid molecules and simultaneous determination of the base sequence of a plurality of nucleic acid molecules based on the time-dependent change in the fluorescence of the nucleic acid molecules or/and of the cleaved nucleotide building blocks caused by the cleavage of nucleotide building blocks wherein the fluorescence is produced by beaming light into the support and generating an evanescent excitation field by internal reflection at the support surface in the area of the immobilized nucleic acid molecules.

2. Method as claimed in claim 1, wherein a support made of glass, plastics, quartz or a composite containing one or more of these materials is used.

3. Method as claimed in claim 1, wherein a total internal reflection is generated at the support surface.

4. Method as claimed in claim 1, wherein the nucleic acid molecules are labelled in such a manner that at least 50% of all nucleotide building blocks of one base type carry a fluorescent marker group.

5. Method as claimed in claim 4, wherein essentially all nucleotide building blocks of one base type carry a fluorescent marker group.

6. Method as claimed in claim 1, wherein individual nucleotide building blocks are cleaved by an exonuclease.

7. Method as claimed in claim 6, wherein T7 DNA polymerase, E. coli exonuclease I or E. coli exonuclease III are used.

8. Method as claimed in claim 1, wherein the evanescent field is generated by beaming in light using a widened laser.

9. Method as claimed in claim 1, wherein the evanescent field is generated at multiple areas on the support by irradiating it with multiple laser beams or/and by means of multiple internal reflections.

10. Method as claimed in claim 1, wherein the determination of the base sequence comprises a detection of the fluorescence emission of a plurality of nucleic acid strands by a detection matrix.

11. Method as claimed in claim 10, wherein a CCD camera or an avalanche photodiode matrix is used.

12. Method as claimed in claim 1, wherein the fluorescence excitation and detection of all examined nucleic acid strands is carried out in parallel.

13. Method as claimed in claim 1, wherein the fluorescence excitation and detection is carried out in several steps and in each case on a portion of the nucleic acid strands to be examined.

14. Method as claimed in claim 1, wherein a convection flow away from the support is generated during the determination.

15. Method as claimed in claim 1, wherein the fluorescent marker groups are at least partially quenched when they are incorporated into the nucleic acid strands and the fluorescence intensity is increased after cleavage.

16. Device for sequencing nucleic acids comprising:

(a) an at least partially optically transparent support comprising a multitude of nucleic acid molecules immobilized thereon where the nucleic acid molecules are present in a single-stranded form and carry several fluorescent marker groups,

(b) a reaction space for the progressive cleavage of individual nucleotide building blocks from the immobilized nucleic acid molecules,

(c) means for exciting fluorescence by beaming light into the support and generating an evanescent excitation field by internal reflection at the support surface in the area of the immobilized nucleic acid molecules and

(d) means for simultaneously determining the base sequence of a plurality of nucleic acid molecules based on the time-dependent change in fluorescence of the nucleic acid molecules or/and of the cleaved nucleotide building blocks caused by cleavage of nucleotide building blocks.

17. (canceled)