Methods for Analyzing Minute Cellular Nucleic Acids

1 . A method for analyzing cellular nucleic acid, the method comprising the steps of:

capturing RNA from a lysed cell onto a substrate;

producing a cDNA/RNA duplex;

removing the RNA from the cDNA/RNA duplex;

priming the cDNA to produce a primer/cDNA duplex;

exposing the primer/cDNA duplex to at least one detectably labeled nucleotide in the presence of a polymerase capable of catalyzing addition of the nucleotide to the primer/cDNA duplex;

detecting incorporation of the nucleotide into the primer portion; and

repeating the exposing and detecting steps at least once.

2 . The method according to claim 1 , wherein prior to the capturing step, the method further comprises lysing a cell to release RNA from that cell.

3 . The method according to claim 1 , wherein capturing comprises:

adding a poly(A) tail to the RNA; and

hybridizing the poly(A) tailed RNA to poly(T) primers that are attached to the substrate.

4 . The method according to claim 3 , wherein producing the cDNA/RNA duplex comprises: performing a sequencing reaction on the poly(A) tailed RNA that is hybridized to the poly(T) primer, thereby producing the cDNA/RNA duplex.

5 . The method according to claim 4 , wherein the sequencing reaction is a sequencing-by-synthesis reaction.

6 . The method according to claim 1 , wherein priming comprises:

determining the sequence of the cDNA;

synthesizing a primer that corresponds to a portion of the cDNA; and

hybridizing the primer to the corresponding portion of the cDNA.

7 . The method according to claim 1 , wherein priming comprises:

adding a poly(G) tail to a 3′ end of the cDNA; and

hybridizing a poly(C) primer to the poly(G) tail.

8 . The method according to claim 7 , wherein prior to the exposing step, the method further comprises: adding dCTP to the primer/cDNA duplex to fill remaining unpaired poly(G) nucleotides on the poly(G) tail of the cDNA, thereby ensuring alignment of the poly(C) primer with the poly(G) tail.

9 . The method according to claim 8 , further comprising: exposing the primer/cDNA duplex to locked nucleic acids.

10 . The method according to claim 3 , wherein prior to the exposing step, the method further comprises: adding dATP in order to fill poly(T) primers on the surface of the substrate that did not pair with an RNA.

11 . The method according to claim 1 , further comprising: determining a sequence of the cDNA based upon the order of incorporation of the labeled nucleotides.

12 . The method according to claim 1 , further comprising: removing unincorporated nucleotide and polymerase in all or some repetitions of the exposing and detecting steps.

13 . The method according to claim 1 , further comprising: neutralizing the label on the labeled nucleotide after the detecting step.

14 . The method according to claim 1 , further comprising:

removing a complementary strand of the cDNA; and

resequencing the cDNA at least once.

15 . The method according to claim 1 , wherein at least a portion of the primer/cDNA duplexes are individually optically resolvable.

16 . The method according to claim 1 , wherein the nucleotide is a non-native nucleotide.

17 . The method according to claim 1 , wherein the detectably labeled nucleotide is an optically labeled nucleotide.

18 . The method according to claim 17 , wherein the optical label is a fluorescent label.

19 . The method according to claim 18 , wherein the fluorescent label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, and conjugated multi-dyes.

20 . The method according to claim 1 , wherein the lysed cells are selected from the group consisting of bacterial cells, fungi cells, and human cells.

21 . The method according to claim 20 , wherein the human cells are cancer cells.