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Nucleic acid sequencing method based on fluorescence quenching

A technology of fluorescence quenching and nucleic acid sequencing, applied in the field of biomedicine, can solve the problems of large DNA damage, high cost and toxicity, and achieve the effect of simple and effective implementation.

Inactive Publication Date: 2010-06-02
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Lu Zuhong et al proposed to prepare a high-density single-molecule multi-copy random whole-genome DNA sequencing template array for the whole genome through single-molecule multi-copy whole-genome amplification [1] , by immobilizing the rolling circle product on the carrier, on the basis of which, on-sheet extension sequencing is performed, each extension of a base has a certain fluorescence intensity, when the fluorescence intensity accumulates to a certain level, it is necessary to quench the fluorescein before further extension, and It is most suitable when no light is needed. There are foreign literatures that use laser to irradiate diphenyl iodine chloride (DPI) to quench fluorescence and then extend [2] , but the damage to DNA is relatively large, DPI drugs are expensive, toxic, and the way of light has certain limitations on high-throughput gene chip sequencing

Method used

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  • Nucleic acid sequencing method based on fluorescence quenching
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Embodiment Construction

[0022] 1. Template preparation: by forming a sequence of rolling circles

[0023] 5’-P-TAGCTAGAATCAAAAATGTTGAGTACGACGAATCTGTATGCTAATGCGGCGTGATGTATTATGCGTATAGAAATAATACAGA-3’ and

[0024] 5’-ACCTTTATGTCAACATTTTTGATTCTAGCTATCTGTATTATTTCACCTAGCTT-3’

[0025] The concentration is 10um and 4ul each are mixed and denatured, and the ring is formed after natural renaturation. The ligation is performed by using T4 ligase. Add probe Acry-probe(100um) 0.3ul(E3: 5’-Acry-(T) 10 ATTAGCATACAGATTCGTCGTACT-3'), through denaturation, natural renaturation and hybridization. Then add 100×BSA, dNTP, Bst enzyme, 40 degrees 30 hours rolling circle.

[0026] The rolling ring product is dissolved with acrylamide glue and fixed on the glass slide modified with acrylamide glue.

[0027] 2. Detection: Hybridize 1uM Cy5-probe (5’-Cy5-GCGGCGTGATGTA) with the fixed product [3] . After scanning, a clear image with high fluorescence intensity can be obtained.

[0028] 3. Quenching: Use quenching agent (H 2 O 2 ) Afte...

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Abstract

The invention relates to a nucleic acid sequence detecting method based on fluorescence quenching. The method includes the steps of: firstly, sequence detecting template preparation: the product of anucleic acid fragment to be detected comprises deoxyribonucleic acid, ribonucleic acid or a nucleic acid sequence enlarged product, the cloned product comprises deoxyribonucleic acid and ribonucleic acid are fixed on carrying agent, secondly, extension in the carrying agent: the mixed fluid of reaction buffer fluid, polymerase and fluorescence marked dNTP are used to perform extension, thirdly, fluorescence quenching: fluorescence quenching agent is used to perform fluorescence quenching, fourthly, extension in the carrying agent: the mixed fluid of the reaction buffer fluid, the polymerase and the fluorescence marked dNTP are used to perform extension, fifthly, image recognition: characteristic point extraction is performed to the obtained extended images, the noise point is eliminated torealize analysis to the chip, or other commercialized software is directly used to perform image processing. The invention combines the nucleic acid micro array chip technology, and can perform large-scale nucleic acid sequencing quickly in high flux and at low cost.

Description

Technical field [0001] The invention is a nucleic acid sequencing method based on fluorescence quenching, and belongs to a genome sequencing method in the field of biomedicine. Background technique [0002] At present, it takes tens of millions of dollars and half a year of work to complete the sequencing of the entire mammalian genome. The cost of DNA sequencing is decreasing by half every two years, but the current general DNA sequencing methods are still costly and time-consuming, far from meeting the requirements of life science and medical development, and also greatly restricting the DNA sequencing market development of. People urgently need to develop fast and cheap individualized genetic information detection technology. [0003] Lu Zuhong et al. proposed to prepare a high-density single-molecule multi-copy random whole-genome DNA sequencing template array for the whole genome through single-molecule multi-copy whole-genome amplification. [1] , By fixing the rolling circl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 高力吕华肖鹏峰钱正瑛陆祖宏
Owner SOUTHEAST UNIV
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