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

A technology of two nucleotides and nucleotides, which is applied to the real-time synthetic DNA sequencing of two nucleotides, realizes the field of high-throughput determination of nucleic acid sequences, and can solve the problems such as the influence of sequencing length.

Inactive Publication Date: 2012-08-15
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 comme

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

The invention discloses a decoding and sequencing method by real-time synthesis of two nucleotides into deoxyribonucleic acid (DNA). Single-sequencing reactions are performed on two different nucleotides of X and Y simultaneously and a base sequence fragment code of 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 sequencing comprises two sets of sequencing reactions on the same template; and in either set of sequencing, a cycle that two different nucleotides synthesize the sequencing reaction simultaneously is performed on deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP) and deoxy-thymidine triphosphate (dTTP) containing four nucleotides in the mode that each nucleotide is used once in a cycle. After a plurality of sequencing reactions, information of a plurality of XYn ranked according to a sequencing order by the first set is obtained. When the set of sequencing reactions is completed, denaturation is performed to eliminate the extended strand of a sequencing primer and the sequencing primer is re-crossed to perform the second set of sequencing reactions; information of a plurality of XYn ranked by the second sequencing reaction is obtained; the specific base sequence of nucleic acid fragment to be detected is assembled by decoding the information of a plurality of XYn ranked according to a sequence order by the two sets.

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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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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