Nanogrid Rolling Circle DNA Sequencing

Abstract

The present invention relates to methods for sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size, including synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region, immobilizing the concatemer to the specific region to make an immobilized concatemer, and sequencing the immobilized concatemer.

What is claimed: 
     
         1 . A method of sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size comprising:
 synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region;   immobilizing the concatemer to the specific region to make an immobilized concatemer; and   sequencing the immobilized concatemer.   
     
     
         2 . The method of  claim 1 , wherein the concatemer has a cross-sectional diameter of at least 200 nanometers. 
     
     
         3 . The method of  claim 1 , wherein the concatemer has a cross-sectional diameter of at least 300 nanometers. 
     
     
         4 . The method of  claim 1 , wherein the polynucleotide is DNA. 
     
     
         5 . The method of  claim 1 , wherein one concatemer is immobilized at the specific region. 
     
     
         6 . The method of  claim 1 , wherein the array has at least 100 specific regions. 
     
     
         7 . The method of  claim 6 , wherein one concatemer is immobilized at each of the specific regions. 
     
     
         8 . The method of  claim 6 , wherein at least 90 of the specific regions each contains one immobilized concatemer. 
     
     
         9 . The method of  claim 1 , wherein the array has at least 1000 specific regions. 
     
     
         10 . The method of  claim 1 , wherein the array has at least 10,000 specific regions. 
     
     
         11 . The method of  claim 1 , wherein sequencing the immobilized concatemer is performed by fluorescent in situ sequencing. 
     
     
         12 . The method of  claim 1 , wherein the array is a nanoarray. 
     
     
         13 . The method of  claim 1 , wherein optical magnification is used to sequence the immobilized concatemer. 
     
     
         14 . The method of  claim 1 , wherein immobilization is performed by hybridization. 
     
     
         15 . The method of  claim 1 , wherein immobilization is performed by an interaction selected from the group consisting of biotin-avidin capture, biotin-streptavidin capture, NHS-ester capture, thioether linkage, static charge interactions and van der Waals forces. 
     
     
         16 . A method of sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size comprising:
 synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter greater than the diameter of a specific region;   immobilizing the concatemer to the specific region to make an immobilized concatemer;   clonally amplifying the immobilized concatemer to make a clonally amplified concatemer; and   sequencing the clonally amplified concatemer.   
     
     
         17 . The method of  claim 16 , wherein clonally amplifying is performed by a method selected from the group consisting of rolling circle amplification, multiple displacement amplification, thermophilic helicase-dependent amplification and bridge PCR. 
     
     
         18 . The method of  claim 17 , wherein rolling circle amplification is selected from the group consisting of hyperbranched rolling circle amplification, padlock probe rolling circle amplification and linear rolling circle amplification. 
     
     
         19 . A method of sequencing a polynucleotide immobilized on an array having a plurality of specific regions each having a defined diameter size of less than 300 nanometers comprising:
 synthesizing a concatemer of a polynucleotide by rolling circle amplification, wherein the concatemer has a cross-sectional diameter of greater than 300 nanometers;   immobilizing the concatemer to the specific region to make an immobilized concatemer; and   sequencing the immobilized concatemer.