Sequencing of surface immobilized polymers utilizing microfluorescence detection

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.

What is claimed is:  
     
         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.