Nucleic acid sequencing method of nucleoside non-labeled FRET

Abstract

The invention discloses a nucleic acid sequencing method of nucleoside non-labeled FRET. The method comprises the following steps: labeling FRET donor and acceptor fluorophores on DNA polymerase or a connector, immobilizing a nucleic acid molecule to be detected, incubating and conjugating the nucleic acid molecule to be detected with the DNA polymerase, and continuously and circularly inputting four sequencing reaction systems in sequence to participate in synthesis of a nucleic acid complementary chain to be detected. According to the method, FRET signal change is caused by spatial distance change between donor-acceptor molecule pairs during base synthesis to determine synthesis, synthesis is combined with input dNTP type to obtain base type corresponding to the reaction, and composition of a nucleic acid sequence is determined through the arrangement of the base types obtained by multiple reactions. The method has low requirements on nucleic acid concentration and total amount of a sample, is suitable for rapid detection in a laboratory, can bring higher sequencing accuracy through a dNTP non-labeling strategy, and can measure FRET signals at a single-molecule level, monoclonal molecular clusters, and single-molecule multiple copies.

Description

technical field

[0001] The invention relates to a high-throughput sequencing method, in particular to a nucleic acid sequencing method for nucleoside non-labeled FRET. Background technique

[0002] FRET (Fluorescence Resonance Energy Transfer) is an energy transfer phenomenon between two fluorescent molecules that are close (1-10nm). The emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore to a certain extent, the excitation energy of the donor can induce the acceptor to emit fluorescence, and the FRET efficiency is inversely proportional to the sixth power of the distance between the donor and the acceptor. Therefore extremely sensitive. In biological research, it is often used to identify the internal conformational changes of proteins and nucleic acid molecules and the interaction between molecules, especially to study the process of nucleic acid chain synthesis by using the characteristic that FRET efficiency is in...

Examples

Embodiment 1

[0032] Preparation and application of microwell arrays: PDMS (polydimethylsiloxane) chips containing countless microwells (2μm×2μm×2μm) were prepared by soft lithography, with more than 500,000 microwells on each chip. And the known sequence is chemically modified on the surface of the chip as a linker to capture the nucleic acid molecule to be tested (6-8 bases of the known sequence are complementary to the linker of the subsequent library construction), and the known sequence is also used as a primer for the synthesis of the complementary strand. The donor (Alexa Fluor 647) and acceptor (Alexa Fluor 555) fluorophores can either be modified at the 5' end of the known sequence, the other can be modified on the DNA polymerase, or both can be modified on the DNA polymerase. Design the nucleic acid sequence according to the known sequence on the chip surface and the A tail added by PCR operation, and complete the connection on the unknown nucleic acid molecule to be tested by DNA ...

Embodiment 2

[0039] On the basis of the sequencing platform in Example 1, a PDMS (polydimethylsiloxane) chip containing micropores of 2 μm×2 μm×2 μm was prepared by soft lithography, with more than 500,000 microwells on each chip, and The known sequence (TCGCCTAT) is chemically modified on the surface of the chip as an adapter to capture the nucleic acid molecule to be tested. Each microwell is modified with countless known sequences, and AlexaFluor 647 is modified at the 5' end of the known sequence. Use the genomic DNA of GM12878 to amplify the HLA-A gene fragment (172bp, NCBI Homo sapiens Gene ID: 3105), 50μL system: Premix Ex Taq HS 25μL, DNA 50ng, forward and reverse primers 0.4μM each (forward primer: 5′ -GGATTACATCGCCCTGAAC-3'; reverse primer: 5'-CGTCTCCTTCCCGTTCTC-3-3'), less than 50 μL was filled with enzyme-free water. The amplification program was: 95°C for 5min; (95°C for 30s, 60°C for 20s, 72°C for 30s) 30 cycles; 72°C for 5min; 4°C for storage. The PCR product is designed wi...

Embodiment 3

[0045]On the basis of the sequencing platform in Example 1, a PDMS (polydimethylsiloxane) chip containing numerous micropores was prepared by soft lithography, each micropore has a diameter of 1 μm and a depth of 2 μm. Contains millions of microwells, and chemically modify known sequences (TCGCCTAT) on the surface of the chip as adapters to capture nucleic acid molecules to be tested. Each microwell is modified with countless known sequences, and the known sequences are also synthesized as complementary strands primers. Use the genomic DNA of GM12878 to amplify the HLA-A gene fragment (172bp), 50μL system: Premix Ex Taq HS 25μL, DNA 50ng, forward and reverse primers 0.4μM each (forward primer: 5′-GGATTACATCGCCCTGAAC-3′; reverse Primer: 5′-CGTCTCCTTCCCGTTCTC-3-3′), less than 50 μL was filled with enzyme-free water. The amplification program was: 95°C for 5min; (95°C for 30s, 60°C for 20s, 72°C for 30s) 30 cycles; 72°C for 5min; 4°C for storage. The nucleic acid sequence (5'-T...

technical field

[0001] The invention relates to a high-throughput sequencing method, in particular to a nucleic acid sequencing method for nucleoside non-labeled FRET. Background technique

[0002] FRET (Fluorescence Resonance Energy Transfer) is an energy transfer phenomenon between two fluorescent molecules that are close (1-10nm). The emission spectrum of the donor fluorophore overlaps with the excitation spectrum of the acceptor fluorophore to a certain extent, the excitation energy of the donor can induce the acceptor to emit fluorescence, and the FRET efficiency is inversely proportional to the sixth power of the distance between the donor and the acceptor. Therefore extremely sensitive. In biological research, it is often used to identify the internal conformational changes of proteins and nucleic acid molecules and the interaction between molecules, especially to study the process of nucleic acid chain synthesis by using the characteristic that FRET efficiency is in...

Examples

Embodiment 1

[0032] Preparation and application of microwell arrays: PDMS (polydimethylsiloxane) chips containing countless microwells (2μm×2μm×2μm) were prepared by soft lithography, with more than 500,000 microwells on each chip. And the known sequence is chemically modified on the surface of the chip as a linker to capture the nucleic acid molecule to be tested (6-8 bases of the known sequence are complementary to the linker of the subsequent library construction), and the known sequence is also used as a primer for the synthesis of the complementary strand. The donor (Alexa Fluor 647) and acceptor (Alexa Fluor 555) fluorophores can either be modified at the 5' end of the known sequence, the other can be modified on the DNA polymerase, or both can be modified on the DNA polymerase. Design the nucleic acid sequence according to the known sequence on the chip surface and the A tail added by PCR operation, and complete the connection on the unknown nucleic acid molecule to be tested by DNA ...

Embodiment 2

[0039] On the basis of the sequencing platform in Example 1, a PDMS (polydimethylsiloxane) chip containing micropores of 2 μm×2 μm×2 μm was prepared by soft lithography, with more than 500,000 microwells on each chip, and The known sequence (TCGCCTAT) is chemically modified on the surface of the chip as an adapter to capture the nucleic acid molecule to be tested. Each microwell is modified with countless known sequences, and AlexaFluor 647 is modified at the 5' end of the known sequence. Use the genomic DNA of GM12878 to amplify the HLA-A gene fragment (172bp, NCBI Homo sapiens Gene ID: 3105), 50μL system: Premix Ex Taq HS 25μL, DNA 50ng, forward and reverse primers 0.4μM each (forward primer: 5′ -GGATTACATCGCCCTGAAC-3'; reverse primer: 5'-CGTCTCCTTCCCGTTCTC-3-3'), less than 50 μL was filled with enzyme-free water. The amplification program was: 95°C for 5min; (95°C for 30s, 60°C for 20s, 72°C for 30s) 30 cycles; 72°C for 5min; 4°C for storage. The PCR product is designed wi...

Embodiment 3

[0045]On the basis of the sequencing platform in Example 1, a PDMS (polydimethylsiloxane) chip containing numerous micropores was prepared by soft lithography, each micropore has a diameter of 1 μm and a depth of 2 μm. Contains millions of microwells, and chemically modify known sequences (TCGCCTAT) on the surface of the chip as adapters to capture nucleic acid molecules to be tested. Each microwell is modified with countless known sequences, and the known sequences are also synthesized as complementary strands primers. Use the genomic DNA of GM12878 to amplify the HLA-A gene fragment (172bp), 50μL system: Premix Ex Taq HS 25μL, DNA 50ng, forward and reverse primers 0.4μM each (forward primer: 5′-GGATTACATCGCCCTGAAC-3′; reverse Primer: 5′-CGTCTCCTTCCCGTTCTC-3-3′), less than 50 μL was filled with enzyme-free water. The amplification program was: 95°C for 5min; (95°C for 30s, 60°C for 20s, 72°C for 30s) 30 cycles; 72°C for 5min; 4°C for storage. The nucleic acid sequence (5'-T...

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