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Methods and systems for direct sequencing of single DNA molecules

a single dna and dna technology, applied in the field of methods and systems for direct sequencing of single dna molecules, can solve the problem of elusive $1000 genome technology, and achieve the effect of rapid dna sequencing

Inactive Publication Date: 2011-12-01
RGT UNIV OF CALIFORNIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]The present invention provides compositions, methods, kits, and systems for rapid DNA sequencing. In some embodiments, sensors are engineered onto the surface of a polymerase molecule to monitor subtle, yet distinct, conformational changes that accompany the incorporation of each base type. Movement of one to tens of angstroms by the polymerase can be measured precisely with the Förster resonance energy transfer (FRET) technique. Multiple FRET pairs (or networks) placed at strategic residues on the polymerase can be used to monitor conformational changes in real time (10 times faster than the rate of DNA synthesis). The sensors can provide multi-parametric information about the dynamic structure of the polymerase, which in turn can provide a unique signature for each base type incorporated. Chemical modifications such as methylation on the template DNA can also be detected according to the disclosed methods.

Problems solved by technology

Despite the great progress that has been made, the $1000 genome technology remains elusive.

Method used

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  • Methods and systems for direct sequencing of single DNA molecules
  • Methods and systems for direct sequencing of single DNA molecules
  • Methods and systems for direct sequencing of single DNA molecules

Examples

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example 1

Design of Labeled Phi-29 DNA Polymerase

[0202]For the sake of illustration, we describe our systematic examination of the crystals structures of phi-29 DNA polymerase complexed with various substrates. We used a genetically engineered exonuclease-deficient phi-29 DNA polymerase (Berman et al., EMBO J, 26:3494-3505 (2007). The mutations involved in eliminating exonuclease activity do not affect the active site, or adjacent sites on the finger, thumb, and palm domains.

[0203]One of skill will appreciate that the sites disclosed for labeling phi-29 DNA polymerase can be applied to other DNA polymerases. As explained above, the structures of DNA polymerases are well-conserved. Thus, through optimal structural alignment (alignment of amino acids present in particular structural positions), the positions disclosed herein can be ascertained for a broad range of polymerases.

[0204]We selected residues on the finger subdomain as candidate labeling sites for the fluorescence donor and some on th...

example 2

In Vitro Translation of a Labeled DNA Polymerase

[0210]We have designed an efficient method for engineering labeled DNA polymerases with multiple labels positioned at designated residues. The general concept is illustrated in FIG. 6. For simplicity, the solid lines separating each named amino acid represent additional amino acids that are not shown.

[0211]The DNA polymerase coding sequence is cloned into a vector. The vector also includes regulatory sequences necessary for transcription (e.g. T7 promoter), translation initiation (ribosomal binding site—RBS, and start codon—ATG), and termination (stop codon—UAG). The codons encoding the targeted residues for labeling are mutated to a codon encoding a cysteine residue (TGC) using standard molecular biology methods.

[0212]The mRNA molecules are captured on a solid support by hybridization of a sequence at one end of the RNA molecules to a complementary oligonucleotide or PNA (peptide nucleic acid) immobilized on the solid support. The mRN...

example 3

READS Technology Using Immobilized DNA Polymerase

[0221]DNA polymerases are labeled as described above, and immobilized on glass coverslips. The surface of a glass coverslip is derivatized with a streptavidin. The glass coverslip is first cleaned with the RCA protocol, derivatized with amine group with aminoalkyl (e.g. gamma-aminopropyl) triethoxysilane, and then functionalized with biotin with NHS ester-PEG-biotin (e.g. NHS ester-PEG 5000-Biotin). The biotinylated surface can be patterned into highly-ordered arrays with feature size and spacing optimal for assembly of single molecular arrays and fluorescent imaging efficiency.

[0222]The biotinylated coverslip is then assembled into a flowcell for further functionalization with streptavidin. The biotinylated surface is functionalized with streptavidin by incubating the glass coverslip with a streptavidin solution, e.g. 1 μM streptavidin in a buffer solution such as phosphate buffer saline (PBS, 137 mM NaCl, 2.7 mM KCl, 10 mM sodium ph...

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Abstract

The invention provides improved methods for sequencing nucleic acids, e.g., for medical applications and biomedical research. The disclosed methods can be applied to rapid personalized medicine, genetic diagnosis, pathogen identification, and sequencing species genomes.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]The present application claims priority to U.S. Provisional Appl. No. 61 / 121,809, filed Dec. 11, 2008, incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]Through massive parallelization and miniaturization, the throughput of DNA sequencing has been increased tremendously while the cost of sequencing has been reduced by several orders of magnitude compared to the conventional gel or capillary-based sequencers using the Sanger dideoxy sequencing method. Several other emerging sequencing platforms can potentially increase the throughput and reduce the cost of DNA sequencing even further by another two orders of magnitude, promising to give us the so-called $1000 genome sequencing technology (Rothberg, J. M. and Leamon, J. H., Nat Biotechnol, 26; 1117-1124 (2008); Schloss, J. A., Nat Biotechnol, 26:1113-1115 (2008); Shendure, J. and Ji, H., Nat Biotechnol, 26:1135-1145 (2008)).[0003]The possibility of $1000 gen...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N9/96C12M1/34C12Q1/68
CPCC12Q1/6869C12N9/1252C12Q2565/101C12Q2565/133
Inventor HUANG, XIAOHUA
Owner RGT UNIV OF CALIFORNIA
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