High speed parallel molecular nucleic acid sequencing

Abstract

A method and device is disclosed for high speed, automated sequencing of nucleic acid molecules. A nucleic acid molecule to be sequenced is exposed to a polymerase in the presence of nucleotides which are to be incorporated into a complementary nucleic acid strand. The polymerase carries a donor fluorophore, and each type of nucleotide (e.g. A, T/U, C and G) carries a distinguishable acceptor fluorophore characteristic of the particular type of nucleotide. As the polymerase incorporates individual nucleic acid molecules into a complementary strand, a laser continuously irradiates the donor fluorophore, at a wavelength that causes it to emit an emission signal (but the laser wavelength does not stimulate the acceptor fluorophore). In particular embodiments, no laser is needed if the donor fluorophore is a luminescent molecule or is stimulated by one. The emission signal from the polymerase is capable of stimulating any of the donor fluorophores (but not acceptor fluorophores), so that as a nucleotide is added by the polymerase, the acceptor fluorophore emits a signal associated with the type of nucleotide added to the complementary strand. The series of emission signals from the acceptor fluorophores is detected, and correlated with a sequence of nucleotides that correspond to the sequence of emission signals.

1 . A method for high speed parallel molecular nucleic acid sequencing, comprising:
 (a) attaching a single nucleic acid molecule to a substrate;   (b) adding an oligonucleotide primer under conditions where the primer anneals to the nucleic acid molecule;   (c) adding a polymerase and nucleotides, wherein the polymerase and nucleotides are labeled with a fluorophore, and wherein each type of nucleotide comprises a different fluorophore that emits a unique emission signal corresponding to addition of that type of nucleotide as the nucleotide is incorporated into a complementary nucleic acid molecule by the polymerase; and   (d) detecting the unique emission signal.   
     
     
         2 . The method of  claim 1 , wherein the unique emission signal is converted into a signal for a specific nucleotide in a nucleic acid sequence. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid molecule is DNA and the polymerase is a DNA or RNA polymerase. 
     
     
         4 . The method of  claim 1 , wherein the nucleic acid molecule is RNA and the polymerase is reverse transcriptase. 
     
     
         5 . The method of  claim 1 , wherein the polymerase is a Klenow fragment of DNA polymerase I. 
     
     
         6 . The method of  claim 1 , wherein the unique emission signal is generated by luminescence resonance energy transfer (LRET) or fluorescent resonance energy transfer (FRET). 
     
     
         7 . The method of  claim 1 , wherein the fluorophore of the polymerase is a donor fluorophore and the fluorophore of each nucleotide is an acceptor fluorophore. 
     
     
         8 . The method of  claim 7 , wherein each of the acceptor fluorophores is stimulated by an emission from the donor fluorophore, and each of the acceptor fluorophores emits a unique emission signal upon stimulation. 
     
     
         9 . The method of  claim 7 , wherein the donor fluorophore is green fluorescent protein (GFP). 
     
     
         10 . The method of  claim 7 , wherein the acceptor fluorophores are BODIPY, fluorescein, rhodamine green, and Oregon green or derivatives thereof. 
     
     
         11 . The method of  claim 7 , wherein the donor fluorophore is excited by a luminescent molecule. 
     
     
         12 . The method of  claim 11 , wherein the donor fluorophore is GFP and the luminescent molecule is aequorin. 
     
     
         13 . The method of  claim 7 , wherein the donor fluorophore is a luminescent molecule. 
     
     
         14 . The method of  claim 11 , wherein the luminescent molecule is aequorin. 
     
     
         15 . The method of  claim 1 , wherein the polymerase is a GFP-polymerase. 
     
     
         16 . The method of  claim 7  wherein the donor fluorophore and one of the acceptor fluorophores comprise a FRET pair selected from the group consisting of GFP mutant H9 and its derivatives, H9-40, tetramethylrhodamine, LISSAMINE™, Texas Red and naphthofluorescein. 
     
     
         17 . The method of  claim 1 , further comprising performing a plurality of sequencing reactions substantially simultaneously, and detecting the signals from the plurality of sequencing reactions. 
     
     
         18 . The method of  claim 17 , wherein a plurality of nucleic acid molecules are fixed directly or indirectly to the substrate in a predetermined pattern, and detecting the signal further comprises correlating the signal with a single nucleic acid molecule corresponding to a predetermined position within that pattern. 
     
     
         19 . The method of  claim 18 , wherein the nucleic acid molecules are fixed to the substrate in the predetermined pattern in channels which have been etched in an orderly array. 
     
     
         20 . The method of  claim 18 , wherein the nucleic acid molecules are fixed to the substrate in the predetermined pattern by micropipetting droplets onto a substrate. 
     
     
         21 . The method of  claim 2 , wherein the unique emission signal is detected with a charged-coupled device (CCD) camera and converted into the nucleic acid sequence. 
     
     
         22 . The method of  claim 1 , wherein the unique emission signals are stored in a computer readable medium. 
     
     
         23 . The method of  claim 1 , wherein the substrate comprises a biocompatible material that is transparent to light. 
     
     
         24 . The method of  claim 1 , wherein the substrate is a glass slide. 
     
     
         25 . The method of  claim 1 , wherein the substrate is a glass microscope slide that is 3 cm long, 1 cm wide and 0.25 cm thick. 
     
     
         26 . The method of  claim 17 , wherein the method comprises sequencing 1000 or more nucleic acid molecules simultaneously. 
     
     
         27 . The method of  claim 1 , wherein the nucleic acid molecule is sequenced at a rate of about 360 bases or more per hour.