System and method for nucleic acid sequencing by polymerase synthesis

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

1 - 143 . (canceled) 
     
     
         144 . A method for genotyping a target nucleic acid, said method comprising:
 providing a primer nucleic acid which complements a region of a target nucleic acid to form a hybridized complement;   immobilizing said target nucleic acid complement to a solid support by means that prevents release;   contacting said target nucleic acid complement with a least one type of labeled nucleotide triphosphate (NTP), wherein each type of NTP is differently labeled; and   detecting the incorporation of said labeled NTP into a single molecule of the primer, to genotype said target nucleic acid.   
     
     
         145 . The method of  claim 144 , wherein said target nucleic acid complement is attached to said solid support by a bonding selected from the group consisting of covalent bonding, ionic boding and hydrophobic bonding. 
     
     
         146 . The method of  claim 145 , wherein said target nucleic acid is attached to the solid support by covalent bonding. 
     
     
         147 . The method of  claim 144 , wherein said target nucleic acid comprises a single nucleotide polymorphism. 
     
     
         148 . The method of  claim 144 , wherein each type of NTP is differently labeled on the γ-phosphate. 
     
     
         149 . The method of  claim 144 , wherein said primer length is at least 10 bp in length. 
     
     
         150 . The method of  claim 144 , wherein said primer length is at least between 15 and 30 bp in length. 
     
     
         151 . The method of  claim 144 , wherein said primer binds to synthetic oligonucleotide adaptors joined to the target nucleic acid. 
     
     
         152 . The method of  claim 144 , wherein said primer is designed to hybridize to a known internal site on the target nucleic acid.