Decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA)

A two-nucleotide, sequencing technology, used in biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve problems such as the impact of sequencing length

Inactive Publication Date: 2013-10-30
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, on the one hand, the price of labeled nucleotide monomers, especially labeled nucleotide monomers with specific cleavage properties, is hundreds of times higher than that of commercially available non-labeled natural nucleotide monomers, and the introduction of cleavage reactions, Changing the structure of natural nucleotides will affect the length of the sequence

Method used

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  • Decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA)
  • Decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA)
  • Decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The two-nucleotide real-time synthetic DNA decoding sequencing method determined the 3'-TAATCAGGTCCCATTTTGGCCTA-5' sequence in the artificially synthesized template containing the 3'-TAATCAGGTCCCATT TTGGCCTA-5' fragment.

[0055] 1. Template preparation: 5’-modified biotin artificially synthesized templates were immobilized with avidin-modified magnetic beads, then the magnetic beads were separated from the liquid, and the artificially synthesized templates immobilized by magnetic beads were used for hybridization with sequencing primers.

[0056] 2. Sequencing primer hybridization: Incubate the designed sequencing primer with the template immobilized by magnetic beads at 75°C for 5 minutes, then cool naturally to room temperature, then separate the magnetic beads from the liquid, and use the template immobilized by magnetic beads for two nucleotides Real-time synthetic DNA sequencing.

[0057] 3. Place the bead-immobilized template in a pyrosequencer for sequencing:

...

Embodiment 2

[0080] Example 2: Two-nucleotide real-time synthetic DNA decoding and sequencing of the Escherichia coli genome

[0081] 1. Preparation of the whole genome template: the Escherichia coli genome is broken into fragments with a size of 100-1000 bp bases by ultrasound, and these fragmented nucleic acid sequences are connected with a pair of universal linkers with known sequences (such as : The sequence of the linker 1 is: CTG CTG TAC CGT ACA GCC TTG GCC G; the sequence of the linker 2 is: CGC TTT CCT CTC TAT GGG CAG TCG GTGA T) for connection and pre-amplification for 10 cycles; The 200-800bp DNA fragment was cut by gel electrophoresis and purified. These 200-800bp DNA fragments were subjected to emulsion parallel PCR reaction with microbeads immobilized with complementary sequences of one of the linkers to amplify the fragmented Escherichia coli genome fragments and denature them to obtain Escherichia coli genome sequencing DNA templates Beads of double-stranded DNA templates a...

Embodiment 3

[0093] Example 3: SNP typing of the 14417 site of the oxidized low-density lipoprotein receptor 1 gene (OLR-1)

[0094] 1. Sample booklet processing: All blood samples were extracted with traditional protein kinase K and phenol / chloroform extraction methods to extract genomic DNA from peripheral blood.

[0095] 2. PCR amplification: 5'-biotin-TACTATCCTTCCCAGCTCCT; 5'-TTTTCAGCAACTTGGCAT-3'. The 50uL PCR amplification system contains 50ng of genomic DNA, 1×PCR buffer, 3mM MgCl2, 250uM dNTPs, 20pmol of two primers, and 2U of Taq DNA polymerase. The amplification conditions were: 5min pre-denaturation at 94°C; 35 cycles of 30s 94°C denaturation, 30s 48°C annealing and 30s 72°C extension; and finally 7min 72°C final extension. The single strand extended by the modified primer in the PCR product has a fragment size of 135 bases, and its sequence is: 5′-biotin-tacta tccttcccag ctcctt gtcc gcaagactgg atctggcatg gagaaaactg ttacctattt tcctcgggct catttaactg ggaaaaSagc caagagaagt gcttgtc...

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Abstract

A decoding and sequencing method by real-time synthesis of two nucleotides into DNA. Single-sequencing reactions are performed on two different nucleotides, namely, X and Y simultaneously, and a base sequence fragment code XYn is obtained according to the quantitative relation between the number of synthesizing nucleotides and the number of the detected molecules which are generated in real time. The entire sequencing comprises performing two sets of sequencing reactions on the same template, wherein each set of sequencing comprises four nucleotides, namely, dATP, dCTP, dGTP, and dTTP, and cycles of sequencing reactions by simultaneous synthesis of two different nucleotides are performed in a manner that each nucleotide is used only once in each cycle. After several sequencing reactions, a set of XYn information ranked in a sequencing order is obtained. When said set of sequencing reactions is completed, denaturation is performed to eliminate an extended strand of a sequencing primer and the sequencing primer is re-crossed to perform a second set of sequencing reactions, and then several XYn information ranked by the second sequencing reaction is obtained; and finally, a specific base sequence of a nucleic acid fragment to be detected is assembled by decoding two sets of XYn information ranked in a sequencing order.

Description

technical field [0001] The invention belongs to the field of biotechnology, and is a method for realizing high-throughput determination of nucleic acid sequences, in particular to a two-nucleotide real-time synthetic DNA sequencing method and its application. Background technique [0002] With the development and completion of the Human Genome Project and various model organism genome projects, human beings have entered the post-gene era, which has had a huge impact on contemporary biological research and medical research, and the related disciplines of molecular biology have been rapidly developed. develop. It will become possible to understand the differences of life, the law of disease occurrence and development, and the interaction between drugs and living organisms at the genetic level. Drastically reducing the cost of DNA sequencing will greatly promote research in life sciences and medicine, and even bring about revolutionary changes. At present, whole-genome DNA se...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/68C12Q1/6869
Inventor 肖鹏峰陆祖宏浦丹钱晓婷
Owner SOUTHEAST UNIV
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