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Title: 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.

Inventors:
; ; ;
Publication Date:
Research Org.:
President and Fellows of Harvard College, Cambridge, MA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1351879
Patent Number(s):
9,624,538
Application Number:
12/120,541
Assignee:
President and Fellows of Harvard College CHO
DOE Contract Number:
FG02-02ER63445
Resource Type:
Patent
Resource Relation:
Patent File Date: 2008 May 14
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Church, George M., Porreca, Gregory J., Shendure, Jay, and Rosenbaum, Abraham Meir. Nanogrid rolling circle DNA sequencing. United States: N. p., 2017. Web.
Church, George M., Porreca, Gregory J., Shendure, Jay, & Rosenbaum, Abraham Meir. Nanogrid rolling circle DNA sequencing. United States.
Church, George M., Porreca, Gregory J., Shendure, Jay, and Rosenbaum, Abraham Meir. Tue . "Nanogrid rolling circle DNA sequencing". United States. doi:. https://www.osti.gov/servlets/purl/1351879.
@article{osti_1351879,
title = {Nanogrid rolling circle DNA sequencing},
author = {Church, George M. and Porreca, Gregory J. and Shendure, Jay and Rosenbaum, Abraham Meir},
abstractNote = {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.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Apr 18 00:00:00 EDT 2017},
month = {Tue Apr 18 00:00:00 EDT 2017}
}

Patent:

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  • Rolling circle amplification (RCA) has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate RCA in solution and in situ to detect gene copy number and single base mutations. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.
  • Cloning of the phi X174 viral origin of replication into phage M13mp8 produced an M13-phi X174 chimera, the DNA of which directed efficient replicative-form----single-strand rolling-circle replication in vitro. This replication assay was performed with purified phi X174-encoded gene A protein, Escherichia coli rep helicase, single-stranded DNA-binding protein, and DNA polymerase III holoenzyme. The nicking of replicative-form I (RFI) DNA by gene A protein was essentially unaffected by the presence of UV lesions in the DNA. However, unwinding of UV-irradiated DNA by the rep helicase was inhibited twofold as compared with unwinding of the unirradiated substrate. UV irradiation of the substratemore » DNA caused a strong inhibition in its ability to direct DNA synthesis. However, even DNA preparations that contained as many as 10 photodimers per molecule still supported the synthesis of progeny full-length single-stranded DNA. The appearance of full-length radiolabeled products implied at least two full rounds of replication, since the first round released the unlabeled plus viral strand of the duplex DNA. Pretreatment of the UV-irradiated DNA substrate with purified pyrimidine dimer endonuclease from Micrococcus luteus, which converted photodimer-containing supercoiled RFI DNA into relaxed, nicked RFII DNA and thus prevented its replication, reduced DNA synthesis by 70%. Analysis of radiolabeled replication products by agarose gel electrophoresis followed by autoradiography revealed that this decrease was due to a reduction in the synthesis of progeny full-length single-stranded DNA. This implies that 70 to 80% of the full-length DNA products produced in this system were synthesized on molecules that carried photodimers.« less
  • A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.
  • Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.
  • This patent describes a process for producing cloned, circular DNA molecules containing shortened target DNA fragments, the fragments derived from a long target DNA segment. The cloned, circular DNA molecules suitable for use in determining the nucleotide sequence of the long target DNA segment. The process consists the steps of: producing, by molecular cloning, double-stranded circular recombinant DNA molecules, each molecule containing vector DNA, a sequencing primer binding site, and a DNA region comprising a long target DNA segment, a first restriction site adjacent to the long target DNA segment adapted to be cut by a first restriction endonuclease inmore » a manner that creates a first terminus on the DNA molecules adjacent the long target DNA segment that is susceptible to digestion by an exonuclease, and a second restriction site located between the first restriction site and the sequencing primer binding site adapted to be cut by a second restriction endonuclease in a manner that creates, without additional terminus blocking or digestion, a second terminus on the DNA molecules that is not susceptible to digestion by an exonuclease; cutting the double-stranded circular recombinant DNA molecules at the first restriction site using a first restriction endonuclease and at the second restriction site using a second restriction endonuclease to form double-stranded linear recombinant DNA molecules having a first terminus that is susceptible to digestion by an exonuclease and a second terminus that is not susceptible to digestion by an exonuclease.« less