Sequencing of surface immobilized polymers utilizing microfluorescence detection

• US 20040029115A9
• Filed: 02/14/2002
• Published: 02/12/2004
• Est. Priority Date: 12/06/1990
• Status: Active Grant

• Current Assignee: Affymax Incorporated
• Original Assignee: Affymax Incorporated
• Inventors: Fodor, Stephen P.A., Dower, William J.
• Granted Patent: US 7,056,666 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C12Q 1/6804 : Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6823 : Release of bound markers
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 : Primer extension
• 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 2537/157 : A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 : fluorescence
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/518 : characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 : characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules

Abstract

Means for simultaneous parallel sequence analysis of a large number of biological polymer macromolecules. Apparatus and methods may use fluorescent labels in repetitive chemistry to determine terminal manomers on solid phase immobilized polymers. Reagents which specifically recognize terminal manomers are used to label polymers at defined positions on a solid substrate.

Claims

1. A method for determining the positions of polymers which terminate with a given monomer, where said polymers are attached to a surface having a plurality of positionally distinct polymers attached thereto, said method comprising the steps of:

labeling a terminal monomer in a monomer type specific manner; and

scanning said surface, thereby determining the positions of said label.

2. A method of claim 1, wherein said polymers are polynucleotides.

3. A method of claim 1, wherein the labeling of the terminal marker comprises incorporation of a labeled terminal monomer selected from the group of nucleotides consisting of adenine, cytidine, guanidine and thymidine.

4. A method for concurrently determining which subset of a plurality of positionally distinct polymers attached to a solid substrate at separable locations terminates with a given terminal subunit, said method comprising the steps of:

mixing said solid substrate with a solution comprising a reagent, which selectively marks positionally distinct polymers which terminate with said given terminal subunit; and

determining with a detector which separable locations are marked, thereby determining which subset of said positionally distinct polymers terminated with said given terminal subunit.

5. A method of claim 4, wherein said solution comprises a reagent which marks said positionally distinct polymer with a fluorescent label moiety.

6. A method of claim 4, wherein said terminal subunit is selected from the group consisting of adenosine, cytosine, guanosine, and thymine.

7. A method for determining which subset of a plurality of primer polynucleotides have a predetermined oligonucleotide, wherein the polynucleotides are complementary to distinctly positioned template strands which are attached to a solid substrate, said method comprising the steps of:

selectively marking said subset of primer polynucleotides having the predetermined oligonucleotide; and

detecting which polynucleotides are marked.

8. A method of claim 7, wherein said oligonucleotide subunit is a single nucleotide.

9. A method of claim 8, wherein the marking comprises elongating said primer with a labeled nucleotide which is complementary to a template.

10. A method of claim 7, wherein the marking step uses a polymerase and a blocked and labeled adenine.

11. A method for concurrently obtaining sequence information on a plurality of polynucleotides by use of a single label detector, said method comprising the steps of:

attaching a plurality of positionally distinct polynucleotides to a solid substrate at separable locations;

labeling said plurality of polynucleotides with a terminal nucleotide specific reagent, said label being detectable using said label detector;

determining whether said specific labeling reagent has labeled each separable location.

12. A method of claim 11, wherein said labeling is performed with reagents which can distinguishably label alternative possible nucleotide monomers.

13. A method of claim 11, wherein at least four replica substrates are produced, each of which is labeled with a specific labeling reagent for adenine, cytosine, guanine, or thymine.

14. A method of claim 11, wherein said labeling and determining steps are performed in succession using reagents specific for each of adenine, cytosine, guanine, and thymine monomers.

15. A method for concurrently obtaining sequence information on a plurality of polynucleotides, said method comprising the steps of:

attaching distinct polynucleotides to a plurality of distinct solid substrates;

labeling said plurality of solid substrates with a terminal nucleotide specific labeling reagent; and

determining whether said specific labeling reagent has labeled each distinct substrate.

16. A method of claim 15, wherein said labeling step is performed in a continuous flow of distinct solid substrates through a reaction solution.

17. A method for simultaneously sequencing a plurality of polymers made up of monomer units, said plurality of polymers attached to a substrate at definable positions, said method comprising the steps of:

mixing said substrate with a reagent which specifically recognizes a terminal monomer, thereby providing identification among various terminal monomer units; and

scanning said substrate to distinguish signals at definable positions on said substrate; and

correlating said signals at defined positions on said substrate to provide sequential series of sequence determinations.

18. A method of claim 17, wherein said plurality of polymers are synthesized by a plurality of separate cell colonies.

19. A method of claim 17, wherein said plurality of polymers are attached to said substrate is by a carbonyl linkage.

20. A method of claim 17, wherein said polymers are polynucleotides.

21. A method of claim 17, wherein said substrate comprises silicon.

22. A method of claim 17, wherein said scanning identifies a fluorescent label.

23. A method of claim 17, wherein said reagent exhibits specificity of removal of terminal monomers.

24. A method of claim 17, wherein said reagent exhibits specificity of labeling of terminal monomers.

25. A method for sequencing a plurality of distinctly positioned polynucleotides attached to a solid substrate comprising the steps of:

hybridizing complementary primers to said plurality of polynucleotides;

elongating a complementary primer hybridized to a polynucleotide by adding a single nucleotide; and

identifying which of said complementary primers have incorporated said nucleotide.

26. A method of claim 25, wherein said elongating step is performed simultaneously on said plurality of polynucleotides linked to said substrate.

27. A method of claim 25, wherein said substrate is a two dimensional surface and said identifying results from a positional determination of said complementary primers incorporating said single defined nucleotide.

28. A method of claim 25, wherein said substrate is a silicon substrate.

29. A method of claim 25, wherein said linking is by photocrosslinking said polynucleotide to said complementary primer, where said primer is attached to said substrate.

30. A method of claim 25, wherein said elongating is catalyzed by a DNA dependent polymerase.

31. A method of claim 25, wherein said single defined nucleotide has a removable blocking moiety to prevent further elongation.

32. A method of claim 31, wherein said blocking moiety is NVOC.

33. A method of claim 25, wherein said single nucleotide has both a blocking moiety and labeling moiety.