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Compositions and methods for sequencing nucleic acids

A nucleic acid sequence, nucleotide sequence technology, applied in the field of nucleotide analogs and their use in nucleic acid sequencing, can solve problems such as expensive and time-consuming

Active Publication Date: 2012-11-14
IND TECH RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although technology has been developed to read a genetic sequence at the nucleotide level, such methods can be time-consuming and extremely expensive

Method used

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  • Compositions and methods for sequencing nucleic acids
  • Compositions and methods for sequencing nucleic acids
  • Compositions and methods for sequencing nucleic acids

Examples

Experimental program
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Embodiment 1

[0203] Here and in the Examples below, exemplary nucleotide analogs may include base B 1 , B 2 and / or group L 1 , L 2 , Q, F, X 1 with X 2 , each having the identity as described herein, represent nucleotide analogs having the structure of Formula I above.

[0204] An exemplary dinucleotide triphosphate analog has the structure shown in Formula II:

[0205] Formula II

[0206] At image 3 A schematic scheme of a single cycle of proofread-dependent sequencing by synthesis using an analog having the structure of Formula II is provided in . A reaction complex comprising a proofreading polymerase, a target (template) strand, and a replicator strand is exposed to excitation light. An incoming nucleotide analog having as base B 1 An adenine that binds to the reactive site of the polymerase and base-pairs with a thymine in the target strand. By the polymerase, the dinucleotide analog is incorporated into the growing strand, whereupon the fluorescent label F is stimulated ...

Embodiment 2

[0208] An exemplary dinucleotide triphosphate analog comprising a fluorescence quenching moiety Q, the dinucleotide triphosphate analog has a structure as shown in Formula III:

[0209] Formula III

Embodiment 3

[0211] An exemplary dinucleotide triphosphate analog having a phosphorothioate that replaces the α-phosphate of the triphosphate chain, thus avoiding the persistent 3' to 5' exonuclease of the polymerase Active, this dinucleotide triphosphate analog has the structure shown in Formula IV:

[0212] right. By the polymerase, the dinucleotide analog is incorporated into the growing strand, whereupon the fluorescent label F is stimulated by the excitation light and emits a signal which is picked up by a detector. as in Figure 4 As shown by the bar at the bottom showing the presence of a signal, the signal remains detectable when the dinucleotide is incorporated into the growing strand by the polymerase and persists until nuclease activity is increased from the polymerase by the polymerase. strand to cut off the unpaired portion of the analog (including the group X, which cannot base pair with the subsequent base in the target strand, and the fluorescent label F). When the lab...

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Abstract

The present invention provides a method for sequencing nucleic acids, comprising the use of nucleotide analogs and a nucleic acid polymerase enzyme or enzyme complex with proofreading activity. The nucleotide analogs may be incorporated into a replicating strand and induce the proofreading activity of the polymerase enzyme, thereby prolonging the duration of a signal associated with nucleotide incorporation, generating remarkable sequencing results and increasing correctness of nucleic acids sequencing.

Description

technical field [0001] The present invention relates to methods for determining the sequence of a nucleic acid. In addition, the present invention relates to nucleotide analogs and their use in nucleic acid sequencing. Background technique [0002] Various techniques and procedures have been developed to obtain genetic information, including broad genetic profiling or identifying patterns of segregated markers in the genetic codes and nucleotides of entire genomes degree of sequencing. For a variety of applications, such as biotechnology, forensic biology, diagnostics, systematic biology, synthetic biology and personal healthcare For basic biological research, knowledge of DNA sequences has become indispensable. The advent of DNA sequencing has dramatically enhanced biological research and discovery. Although techniques have been developed to read a gene sequence at the nucleotide level, such methods can be time consuming and prohibitively expensive. [0003] By moving ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC07H21/04C12Q1/6869C07H21/00C12Q2525/117C12Q2525/119C12Q2565/101
Inventor 邱创汎潘诏智范振业张上嘉傅祖儒
Owner IND TECH RES INST
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