CN111926063A - DNA molecule detection method using 3D bar code - Google Patents
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Abstract
The invention specifically relates to a DNA molecule detection method utilizing a 3D barcode, which belongs to the technical field of gene diagnosis, and specifically comprises the steps of designing a DNA single-chain loop C1 and a DNA single-chain L2 according to a DNA sequence to be detected, hybridizing sequences at two ends of the DNA single-chain L2 with a segment of sequence of the DNA single-chain loop C1, connecting sequence gaps at two ends of the DNA single-chain L2 to form a DNA single-chain double-loop C1C2, hybridizing and connecting with a probe on the 3D barcode, hybridizing the DNA to be detected with the DNA single-chain double-loop C1C2, obtaining part of double-chain DNA with restriction enzyme cutting sites, cutting the double-chain DNA by restriction enzyme to release a topological structure, carrying out solid-phase RCA reaction on the surface of the 3D barcode. The DNA molecule detection method can realize the detection of DNA single base mutation, has high detection sensitivity and strong specificity, and can detect 0.1 percent of mutation at least.
Description
Technical Field
The invention belongs to the field of gene diagnosis, and particularly relates to a DNA molecular detection method of a 3D barcode.
Background
The gene detection is a technology for detecting trace DNA by blood, other body fluids or cells, and is a method for detecting DNA molecular information in cells of a detected person by a specific device after the peripheral venous blood or other tissue cells of the detected person are taken and amplified, and analyzing whether the gene type, the gene defect and the expression function of the gene contained in the cells are normal or not, so that the method can diagnose diseases and can also be used for predicting the risk of the diseases. The current DNA detection methods are mainly ARMS-PCR (amplification refractory mutation system-PCR), ddPCR (droplet digital PCR) or sequencing. These methods are insensitive, complex and time consuming or expensive and require specialized experimental training. Therefore, it is necessary to establish a method for detecting a trace amount of DNA which is inexpensive, easy to detect, and has both high sensitivity and high specificity.
Suspension microarray is an emerging multiplexed parallel detection method that is favored for its ability to extend array alignment, fast reaction kinetics, and multiplexing flexibility. Barcodes, especially multispectral fluorescently encoded barcodes, are one of the main strategies in floating arrays, and have gained very high attention in recent years in terms of energy encoding capability, convenient decoding systems, stable fluorescent signals, and the like. Different barcodes can be programmed by adjusting different ratios of phosphors during the encoding process, such as with fluorescent dyes or Quantum Dots (QDs). Meanwhile, the flow cytometer is a high-throughput, high-sensitivity detection platform having a laser and a detector arranged along a flow channel to decode a barcode and output different fluorescence intensities. When the bar code is applied to the suspension microarray, because a plurality of fluorescent dyes are introduced in the encoding and decoding processes, high background interference exists in the whole detection system, and when biological macromolecular targets such as nucleic acid, antigen or antibody are combined on the bar code, a plurality of signal amplification technologies are required to improve the whole detection sensitivity. Therefore, it is urgently needed to develop a method for detecting trace DNA molecules with high specificity and strong anti-external interference capability.
Disclosure of Invention
In view of the above, the present invention is directed to a method for detecting a DNA molecule using a 3D barcode.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a DNA molecule detection method using 3D barcodes, comprising the following steps:
(1) designing a DNA single-chain loop C1 and a DNA single-chain L2 according to a DNA sequence to be detected, completely hybridizing sequences at two ends of the DNA single-chain L2 with a segment of sequence of the DNA single-chain loop C1, and connecting sequence gaps at two ends of the DNA single-chain L2 by using DNA ligase to form a DNA single-chain double-loop C1C 2; the DNA single strand L2 has a sequence complementary to the DNA to be detected on the intermediate sequence;
(2) a fluorescent dye-encoded carboxylated 3D barcode-coupled probe having a sequence complementary to the DNA single-stranded loop C1;
(3) hybridizing and connecting the DNA single-chain double-ring C1C2 with the complementary sequence of the DNA single-chain ring C1 of the probe in the step (2), hybridizing the DNA to be detected with the complementary sequence of the DNA to be detected on the DNA single-chain double-ring C1C2 to form partial double-chain DNA, wherein the partial double-chain DNA is provided with restriction enzyme cutting sites;
(4) adding restriction enzyme cutting sites of the restriction enzyme in the restriction enzyme cutting step (3), taking a DNA single-chain loop C1 as a template, adding biotin-labeled free base, and performing solid-phase RCA reaction to obtain a biotinylated RCA reaction product;
(5) and adding streptavidin modified quantitative protein to connect with biotinylated RCA reaction product, and finally performing fluorescence decoding.
As one of the preferable technical schemes, the detection method can detect more than one DNA to be detected simultaneously.
As one of the preferable technical schemes, in the step (2), the coupling is coupling by a carbodiimide method.
As one of the preferable technical scheme, in the step (4), the free base is one or more of dCTP, dATP, dGTP or dTTP.
In a preferred embodiment, in step (5), the quantitative protein is phycoerythrin.
As one of the preferable technical proposal, in the step (5), the detection wavelengths used by the fluorescence decoding are 635nm and 532nm respectively.
As one of the preferable technical scheme, in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID NO.1, the nucleotide sequence of the DNA single-chain loop C1 is shown as SEQ ID NO.5, and the nucleotide sequence of the DNA single-chain L2 is shown as SEQ ID NO. 6; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 13.
As one of the preferable technical scheme, in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID NO.2, the nucleotide sequence of the DNA single-chain loop C1 is shown as SEQ ID NO.7, and the nucleotide sequence of the DNA single-chain L2 is shown as SEQ ID NO. 8; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 14.
As one of the preferable technical scheme, in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID NO.3, the nucleotide sequence of the DNA single-stranded loop C1 is shown as SEQ ID NO.9, and the nucleotide sequence of the DNA single-stranded chain L2 is shown as SEQ ID NO. 10; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 15.
One of the preferable technical solutions is characterized in that, in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID No.4, the nucleotide sequence of the single-stranded loop C1 of the DNA is shown as SEQ ID No.11, and the nucleotide sequence of the single-stranded chain L2 of the DNA is shown as SEQ ID No. 12; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 16.
As one of the preferable technical schemes, in the step (4), the restriction enzyme is HpyCH4 IV.
As one of the preferable technical schemes, the primer sequence used in the solid-phase RCA reaction is SEQ ID NO. 17.
As one of the preferable technical proposal, in the step (4), the lowest detection limit value of the DNA to be detected of the detection method is 0.1%.
The invention has the beneficial effects that:
the invention innovatively introduces a DNA interlocking structure on the surface of the 3D barcode, and realizes the discrimination and quantitative detection of various DNAs in the multiplex DNA detection. The DNA interlock structure comprises a detection loop C1 and a recognition loop C2 entangled through a mechanical interlock topology. In the presence of the DNA to be detected, the recognition loop C2 hybridizes with the DNA to be detected to form a restriction endonuclease recognition site, which can be recognized and cleaved by the Restriction Endonuclease (RE) to release the mechanically interlocked topology, and then the detection loop C1 can serve as a solid-phase RCA reaction template to initiate a signal amplification process. In the whole reaction, the restriction enzyme of RE has high specificity to the DNA to be detected, even can detect the mutation of one base, can realize high-sensitivity detection through solid-phase RCA reaction, and can detect 0.1 percent of mutation at least. The 3D bar code can simultaneously identify various DNAs to be detected and carry out quantitative detection, the DNA interlocking structure is coupled on the surface of the 3D bar code, and solid-phase RCA reaction occurs, so that the weakening condition of a detection signal caused by high background noise can be reduced, and the detection sensitivity of the whole reaction is improved. The perfect combination of the high specificity and high sensitivity detection of the DNA interlocking structure and the multiple parallel detection of the 3D bar code can simultaneously realize the qualitative and quantitative detection of various target DNAs, and the detection method has the advantages of high efficiency, time saving, labor saving and economy, and can be widely applied to the gene screening of tumors and genetic diseases.
Drawings
FIG. 1 is a diagram of the synthesis of a single-stranded double-loop DNA, C1C 2;
FIG. 2 is a 3D scattergram showing the fluorescence signals of the barcode encoded by the excitation of red laser (635nm) along the X-axis (635nm/680nm) and Y-axis (635nm/705nm) for the specific detection of the DNA to be detected; the Z axis (532nm/578nm) represents a fluorescent signal generated by exciting phycoerythrin by green (532nm) laser;
FIG. 3 is an electron microscope scan before and after solid-phase RCA reaction of 3D barcodes, wherein a is a 3D barcode of a coupled probe, and b is a 3D barcode of a solid-phase RCA product combined with phycoerythrin;
FIG. 4 is a graph showing the results of sensitivity detection of 4 types of DNA to be detected.
Detailed Description
Hereinafter, preferred embodiments of the present invention will be described in detail by taking the detection of circulating tumor DNA (ctDNA) as an example with reference to the accompanying drawings.
Example 1
DNA molecule detection method using 3D barcode
Preparation of DNA Single-Strand-bicycle C1C2
Searching a ctDNA gene sequence on NCBI, taking 4 segments of the sequence as DNA to be detected, respectively designing 4 DNA single-chain loops C1 and DNA single-chain L2 according to each segment of the DNA sequence to be detected, wherein the nucleotide sequence is shown as follows, the sequence of the bold italic part is a complementary sequence of the single-chain loop C1 and the single-chain L2, and the underlined sequence is a complementary sequence of the single-chain L2 and the DNA to be detected.
DNA-1 to be tested: 5'-GGAGCTGGTGACGTAGGCAAG-3' (SEQ ID NO.1)
DNA-2 to be tested: 5'-GTTGGAGCACGTGGTGTTGGG-3' (SEQ ID NO.2)
DNA-3 to be tested: 5'-AATGATGCACGTCATGGTGGC-3' (SEQ ID NO.3)
DNA-4 to be tested: 5'-AAAATAGGTAATTTGGGTCTA-3' (SEQ ID NO.4)
C1-1：5’-ACTGTAACCATTCTTGTTTCCCAACCCGCCCTACCCAATATCATTTATCTGAATACCGTG-3’(SEQ ID NO.5)
L2-1：5’-PO43-CACGGTATTCAGATTCTCTCTCTCTCTCCTCTTGCCTACGTCACCAGCTCCGGTTCGATCAAGATCTCTCTCTCTCTCTCTCAGAATGGTTACAGT-3’(SEQ ID NO.6)
C1-2：5’-ACTGTAACCATTCTTGTTTCGTATATTCATAGATGCGATATCATTTATCTGAATACCGTG-3’(SEQ ID NO.7)
L2-2：5’-PO43-CACGGTATTCAGTCTCTCTCTCTCTCTCCTCCCAACACCACGTGCTCCAACGGTTCGATCAAGATCTCTCTCTCTCTCTCTCTCAATGGTTACAGT-3’(SEQ ID NO.8)
C1-3：5’-ACTGTAACCATTCTTGTTTCGCATTGCAGAATTGATTATATCATTTATCTGAATACCGTG-3’(SEQ ID NO.9)
L1-3：5’-PO43-CACGGTATTCAGATTCTCTCTCTCTCTCCTGCCACCATGACGTGCATCATTGGTTCGATCAAGATCTCTCTCTCTCTCTCTCAGAATGGTTACAGT-3’(SEQ ID NO.10)
C1-4：5’-ACTGTAACCATTCTTGTTTCGGATATTAGATGCCATTATATCATTTATCTGAATACCGTG-3’(SEQ ID NO.11)
L2-4：5’-PO43-CACGGTATTCAGATAATCTCTCTCTCTCCTTAGACCCAAATTACCTATTTTGGTTCGATCAAGATCTCTCTCTCTCTCTCCAAGAATGGTTACAGT-3’(SEQ ID NO.12)
As shown in FIG. 1, the sequences at both ends of the DNA single strand L2 are hybridized with a sequence of the DNA single-stranded loop C1, and T4 ligase is connected with the sequence gaps at both ends of the DNA single strand L2 to form a DNA single-stranded double loop C1C 2. The hybridization conditions were: 40pmol of the single stranded DNA strand C1 was denatured with 160pmol of the single stranded DNA L2 at 95 ℃ for 5 minutes, held at 65 ℃ for 30 minutes, slowly cooled to 25 ℃ at a rate of 1 ℃/min, added with 1. mu. L T4 DNA ligase, ligated overnight at 16 ℃, added with 1. mu.l of exonuclease (5U/. mu.l) and 2. mu.l of exonuclease (200U/. mu.l), and held at 37 ℃ for 30 minutes. And finally, recovering the DNA single-chain double-ring C1C2 by using a Page gel recovery kit (Shanghai Biotech, B610357), and detecting by using polyacrylamide gel electrophoresis (120V, 50min) to obtain 4 kinds of correctly connected DNA single-chain double-ring C1C 2.
(II) 3D Bar code coupled Probe
A corresponding probe Primer1-4 is designed according to the DNA to be detected.
Primer1：5’-NH2-ATCAATTCTGCAATGATA-3’(SEQ ID NO.13)
Primer2：5’-NH2-ATCGCATCTATGAATATA-3’(SEQ ID NO.14)
Primer3：5’-NH2-ATAATCAATTCTGCAATG-3’(SEQ ID NO.15)
Primer4：5’-NH2-ATCAATTCTGCAATGATA-3’(SEQ ID NO.16)
The carboxylated 3D barcodes 1-4(3D-B) coded by four fluorescent dyes and 1nmol of the probe Primerl-4 with the corresponding serial number are respectively added into 50 μ l of 2- (N-morpholine) ethanesulfonic acid (MES) with the concentration of 0.1mol/L, pH of 4.5 and mixed, and each 3D barcode is 5X 106Then, 2.5. mu.l of 10 g/L1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) working solution was added thereto and incubated for 30 minutes. Then, 2.5. mu.l of EDC was added thereto, and the mixture was incubated for 30 minutes, washed with 1mL of Tween 20 at a mass concentration of 0.02% and 1mL of 1g/L sodium dodecylsulfate, and finally resuspended in 100. mu.l of Tris EDTA buffer (10mM Tris (pH 8.0), 1mM EDTA) to obtain a 3D barcode 1-4(3D-B @ P1-4) solution of the coupled probe using Beckmann CoulterTeZ 2 full-automatic cell analysis counter.
(III) solid-phase RCA reaction
10pmol of each DNA single-stranded bicyclo C1C 21-4 was ligated to 10pmol of 3D-B @ P1-4 of the corresponding sequence number, and the ligation products were mixed together after 30 minutes at 37 ℃.
Experimental groups: 4 kinds of ligation product mixed liquor, to-be-tested DNA-1, 4 kinds of ligation product mixed liquor, to-be-tested DNA-2, 4 kinds of ligation product mixed liquor, to-be-tested DNA-3, 4 kinds of ligation product mixed liquor, to-be-tested DNA-4, 4 kinds of ligation product mixed liquor and to-be-tested DNA1-4 mixed liquor; control group: 4 ligation product mixtures + control DNA (NC, DNA fragment without test DNA sequence)
Each of the test DNA and the control DNA was 10pmol, mixed, and kept at 37 ℃ for 30 minutes, and 1. mu.l of HpyCH4IV was added thereto, kept at 37 ℃ for 30 minutes, and kept at 70 ℃ for 2 minutes to inactivate HpyCH4 IV. To the cleavage product were added 10pmol of primer DP1, 2. mu.l of biotin-dCTP, 2. mu.l of dATP, 2. mu.l of dGTP, 2. mu.l of dTTP, and 1. mu.l of phi29 DNA polymerase, and the total amount was 25. mu.l, and the mixture was kept at 30 ℃ for 60 minutes to obtain a solid-phase RCA product. Finally, 75 μ l of streptavidin-modified phycoerythrin (SA-PE) was added to the solid-phase RCA product, and incubated at 48 ℃ for 15 minutes to obtain a 3D barcode solution of the solid-phase RCA product bound with phycoerythrin. And detecting the coded fluorescence of the 3D barcode and the reported fluorescence on the SA-PE by using an instrument Luminex200, wherein the detection wavelengths are 635nm of red excitation light and 532nm of green excitation light respectively.
DP1：5’-ATGATATTGGGTAGGGCGGGTT-3’(SEQ ID NO.17)
As a result, as shown in fig. 2, four types of 3D barcodes form four separate clusters, each cluster rises along the Z axis only in the presence of corresponding DNA to be detected, and when no DNA to be detected exists, none of the four clusters rises, and the rise height serves as a quantitative index of the DNA to be detected.
And (3) dripping 5uL of the 3D bar code solution of one coupling probe and the 3D bar code solution of the solid-phase RCA product combined phycoerythrin on a dry and clean silicon chip respectively, spraying gold after drying, and observing by using a scanning electron microscope. As shown in FIG. 3, the diameter of the 3D barcode is about 5-6 μm, the surface of the probe-coupled 3D barcode is very smooth (shown as a in FIG. 3), the surface of the phycoerythrin-bound 3D barcode of the solid-phase RCA product is uneven, and many scattered round protrusions (shown as b in FIG. 3) are formed, thereby verifying the successful occurrence of the solid-phase RCA on the surface of the 3D barcode.
Example 2
Detection of sensitivity of DNA to be detected
Mixing the tested DNA1-4 with the control DNA respectively according to the molar mass ratio of 0: 100, 0.1: 99.9, 0.2: 99.8, 0.3: 99.7, 0.4: 99.6, 0.5: 99.5, 0.8: 99.2, 1: 99, 10: 90 and 100: 0 to ensure that the total copy number is 1011copies/mL to form 4 target DNA solutions containing different tested DNA concentrations, wherein the ratio of each tested DNA is 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 10% and 100%. The target DNA solution was examined according to the method of example 1, and as a result, as shown in FIG. 4, DNA-1 to be examined had a significant difference in fluorescence intensity signals between 0.3% and 0.2%, and the fluorescence intensity at a ratio of 0.2% was almost zero as compared with the fluorescence intensity at 0.1% and 0%; while the fluorescence intensity of the DNA-1 ratio of 0.3% is significantly increased, and the fluorescence intensity is continuously increased with the increase of the ratio, whereby it can be concluded that the detection sensitivity of the 3D barcode to DNA-1 is 0.3%. Similarly, the detection sensitivity of DNA-2, 3, 4 was 0.1%, 0.2% and 0.2%, respectively.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.
Sequence listing
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Claims (10)
1. A DNA molecule detection method using 3D barcodes is characterized by comprising the following steps:
(1) designing a DNA single-chain loop C1 and a DNA single-chain L2 according to a DNA sequence to be detected, completely hybridizing sequences at two ends of the DNA single-chain L2 with a segment of sequence of the DNA single-chain loop C1, and connecting sequence gaps at two ends of the DNA single-chain L2 by using DNA ligase to form a DNA single-chain double-loop C1C 2; the DNA single strand L2 has a sequence complementary to the DNA to be detected on the intermediate sequence;
(2) a fluorescent dye-encoded carboxylated 3D barcode-coupled probe having a complementary sequence of a partial DNA single-stranded loop C1;
(3) hybridizing and connecting the DNA single-chain double-ring C1C2 with a complementary sequence of the DNA single-chain ring C1 of the probe in the step (2), hybridizing the DNA to be detected with the complementary sequence of the DNA to be detected on the DNA single-chain double-ring C1C2 to form partial double-chain DNA, wherein the partial double-chain DNA is provided with a restriction enzyme cutting site;
(4) adding restriction enzyme cutting sites of the restriction enzyme in the restriction enzyme cutting step (3), taking a DNA single-chain loop C1 as a template, adding biotin-labeled free base, and performing solid-phase RCA reaction to obtain a biotinylated RCA reaction product;
(5) and adding streptavidin modified quantitative protein to connect with biotinylated RCA reaction product, and finally performing fluorescence decoding.
2. The method for detecting a DNA molecule according to claim 1, wherein the detection method can simultaneously detect more than one DNA to be detected.
3. The method for detecting a DNA molecule according to claim 1 or 2, wherein in the step (4), the free base is one or more of dCTP, dATP, dGTP or dTTP.
4. The DNA molecule detection method of claim 3, wherein in step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID No.1, the nucleotide sequence of the DNA single-stranded loop C1 is shown as SEQ ID No.5, and the nucleotide sequence of the DNA single-strand L2 is shown as SEQ ID No. 6; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 13.
5. The DNA molecule detection method of claim 3, wherein in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID No.2, the nucleotide sequence of the DNA single-stranded loop C1 is shown as SEQ ID No.7, and the nucleotide sequence of the DNA single-strand L2 is shown as SEQ ID No. 8; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 14.
6. The DNA molecule detection method of claim 3, wherein in the step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID No.3, the nucleotide sequence of the DNA single-stranded loop C1 is shown as SEQ ID No.9, and the nucleotide sequence of the DNA single-strand L2 is shown as SEQ ID No. 10; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 15.
7. The DNA molecule detection method of claim 3, wherein in step (1), the nucleotide sequence of the DNA to be detected is shown as SEQ ID No.4, the nucleotide sequence of the DNA single-stranded loop C1 is shown as SEQ ID No.11, and the nucleotide sequence of the DNA single-strand L2 is shown as SEQ ID No. 12; in the step (3), the nucleotide sequence of the probe is shown as SEQ ID NO. 16.
8. The method for detecting a DNA molecule according to any one of claims 4 to 7, wherein in the step (4), the restriction enzyme is HpyCH4 IV.
9. The method for detecting a DNA molecule according to any one of claims 4 to 7, wherein in the step (4), the primer sequence used in the solid-phase RCA reaction is SEQ ID NO. 17.
10. The method for detecting a DNA molecule according to claim 9, wherein the detection method has a minimum detection limit of 0.1% for DNA to be detected.
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Inventor after: Zhao Shuang Inventor after: Yang Sha Inventor after: Chen Ming Inventor after: Sing Kai Inventor after: Zhan Xinyu Inventor after: Tang Xiaoqi Inventor after: Xiao Yao Inventor before: Yang Sha Inventor before: Chen Ming Inventor before: Sing Kai Inventor before: Zhan Xinyu Inventor before: Tang Xiaoqi Inventor before: Xiao Yao Inventor before: Zhao Shuang |