CN112342269B - Method for capturing nucleic acid molecules and application thereof - Google Patents
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Abstract
The application relates to a method for capturing nucleic acid molecules and application thereof. The application discloses a method for capturing nucleic acid molecules, which comprises a first nucleic acid chain and a second nucleic acid chain, wherein the first nucleic acid chain and the second nucleic acid chain are partially matched to form a double-chain region, at least one end of the nucleic acid molecule is a single-chain region. Meanwhile, the application discloses a hybridization complex obtained by the capturing method and application thereof in sequencing.
Description
Technical Field
The present application relates to the field of molecular biology. In particular, the application relates to methods of capturing nucleic acid molecules, and uses thereof.
Background
Sequence capture techniques are selective isolation or enrichment of specific fragments of the genome with probes designed for known specific genomic regions. Common methods of sequence capture are probe capture, PCR capture. Sequence capture by probe capture methods typically uses the complementary nature of the probe to the target sequence to capture the target sequence, which is also a single-stranded sequence.
Second generation sequencing and single molecule sequencing often involve ligation of DNA fragments to the adaptors of specific adaptors to form a sequencing library in terms of library construction. The type of linker directly affects the ligation efficiency, as well as the ratio of available libraries in the sequencing library. Different sequencing platforms require different sequencing linkers, and each platform requires an optimized library building method and used linkers, such as in Illumina library building, and the Y-linker and the buffer linker have different advantages. In the sequencing field, sample processing before on-machine comprises nucleic acid processing, library construction and the like to be further optimized so as to improve the library construction efficiency and simplify the operation flow.
Disclosure of Invention
The application provides a method for capturing target nucleic acid molecules, which can be used for directly capturing double-stranded molecules or single-stranded molecules by utilizing a nucleotide sequence captured by the method, and can be used for reducing the probability of nonspecific hybridization and improving the hybridization efficiency with a target fragment (a first strand).
In a first aspect, the application discloses a method of capturing a nucleic acid molecule comprising a first nucleic acid strand and a second nucleic acid strand which are complementary, said nucleic acid molecule having at least one single-stranded end, at least one of said single-stranded ends being located on said first nucleic acid strand, the method comprising capturing said nucleic acid molecule with a probe, such that said probe and at least a part of said first nucleic acid strand are complementary to each other to obtain a hybridization complex, at least a part of said first nucleic acid strand comprising said single-stranded end.
It will be appreciated that the first and second nucleic acid strands of the nucleic acid molecule are two different strands, or may be one strand, and that when the nucleic acid molecule is one strand the first and second nucleic acid strands are joined together at one end of the double stranded region to form a nucleic acid molecule having a single stranded region at one end.
The single-stranded ends may be located at the 3 'end and the 5' end of the first nucleic acid strand, the primary function of the single-stranded region being complementary pairing with the probe. Optionally, the single-stranded end is located at the 3' end of the first nucleic acid strand. Based on the principle of complementary pairing of nucleic acids, it is known that when a probe is paired with a first nucleic acid strand, the probe can be used as a primer when the single-stranded end is located at the 3' -end of the first nucleic acid strand, and the first nucleic acid strand can be used as a template for PCR amplification.
Meanwhile, it is understood that the sequence probe paired with the first nucleic acid strand contains an oligonucleotide strand, and when the oligonucleotide strand is located at one end of the probe, the other end of the probe may be a nucleotide sequence or other sequences, such as an amino acid sequence, and the other end of the probe may have other modifications, such as amino modifications, fluorescent modifications, and the like.
The choice of the oligonucleotide strand in the probe will take into account all factors such as annealing temperature, nucleotide composition, length and internal secondary structure of the oligonucleotide strand, in one embodiment of the application the length of the oligonucleotide strand is 20-80 nt.
Further, the nucleotide at the 3 '-end of the above-mentioned oligonucleotide chain contains a hydroxyl group (-OH) so that the 3' -end of the oligonucleotide chain can be linked to other nucleotides via phosphodiester bonds.
In one embodiment, the probe is immobilized on a solid medium selected from at least one of glass, plastic, and magnetic beads. The probe is fixed on a solid medium, so that on one hand, the separation and purification of the nucleic acid sequence after the probe captures the nucleic acid sequence are facilitated; alternatively, the capture of the nucleic acid sequence may be followed by other detection or manipulation, such as PCR amplification, sequencing reactions, and the like.
In one embodiment, the above nucleic acid molecule is obtained by ligating a first nucleic acid fragment and a second nucleic acid fragment, both of which are double stranded DNA, the first nucleic acid fragment being of known sequence, the first nucleic acid fragment comprising a first strand and a second strand that are complementary, the single stranded end being located on the first strand.
The nucleic acid fragments may be ligated in a manner selected from blunt end ligation and/or cohesive end ligation. The nucleic acid fragments with flat ends at both ends can be connected after T/A is added to the ends, so that the connection efficiency can be improved and the connection directionality can be ensured.
It will be appreciated that the nucleic acid fragments may be linked by chemical bonds using a phosphodiester linkage but are not limited to a phosphodiester linkage, and that when the two are linked by a phosphodiester linkage, the 5' end of the first strand has a phosphate group. The natural nucleotide contains phosphate groups and hydroxyl groups, and is chemically connected by adopting phosphodiester bonds, so that the connection product does not damage the natural structural characteristics of the nucleotide chain.
In one embodiment, the 3 'end of the second strand is free of hydroxyl groups, such that the 3' end of the second strand cannot be linked to the second nucleic acid fragment via a phosphodiester linkage. With this scheme, the ligation product will take two forms, one in which the second strand is linked to the second nucleic acid fragment via a non-phosphodiester linkage; another form is that the ligation product does not contain a second strand, and when both strands of the ligation product are used as PCR substrates, the second strand cannot be effectively amplified, and if the ligation product is used as a sequencing substrate, detection of invalid data can be reduced, and the amount of valid detection data can be increased.
The two complementary strands in the first nucleic acid fragment are selected, wherein the temperature difference between the two complementary strands is at least 10 ℃ and not higher than 80 ℃, namely, the temperature difference between the two complementary strands is not less than 80 ℃, namely, tm1-Tm2 is not less than 10 ℃ and not less than 90 ℃ and not less than 1 Tm 50 ℃, tm1 is the dissolution temperature of the first strand, and Tm2 is the dissolution temperature of the second strand. The appropriate difference in dissolution temperature is chosen to ensure preferential binding of the first strand to the probe after denaturation annealing. The probe may be complementarily paired with all or part of the sequence of the first strand, and preferably one scheme is that all of the sequence of the first strand is complementarily paired with the probe. It will be appreciated that complementary pairing of the first strand with the probe is to ensure preferential hybridization of the first strand with the probe at the chosen annealing temperature, and thus that mismatched sequences are allowed to exist in the paired sequences of all sequences of the first strand with the probe under conditions which meet the above requirements.
In one embodiment of the present application, tm1 is 71 ℃, and tm2=45.6 ℃. After the dissolution temperature of the two strands is determined, an annealing temperature T, tm2-5 ℃ T1-5 ℃, is selected for hybridization with the probe, and in one embodiment of the application, the annealing temperature is selected to be 55 ℃.
In one embodiment of the present application, the method of capturing nucleic acid molecules of the present application comprises the steps of: ligating the first nucleic acid fragment and the second nucleic acid fragment to obtain a nucleic acid molecule; denaturation to allow the nucleic acid molecules to melt into single strands; rapidly cooling, and rapidly placing the nucleic acid molecule solution containing the melted nucleic acid molecules on ice; hybridization capturing; wherein the first nucleic acid fragment and the second nucleic acid fragment are both double-stranded DNA, the first nucleic acid fragment is a known sequence, the first nucleic acid fragment comprises a first strand and a second strand which are complementary, the single-stranded ends are located on the first strand, the first nucleic acid strand and the second nucleic acid strand, the nucleic acid molecule has at least one single-stranded end, at least one of the single-stranded ends is located on the first nucleic acid strand, wherein the 5 '-end of the first strand has a phosphate group, the 3' -end of the second strand has no hydroxyl group, and all sequences of the first strand are complementarily paired with a probe for capture. The capturing of nucleic acid molecules by this method can greatly improve the capturing efficiency, eliminating the influence of the second strand on capturing.
When the nucleic acid molecule and the probe are complementarily combined, the combination condition of the nucleic acid molecule and the probe, especially the combination efficiency and the combination position, needs to be intuitively judged, so as to judge whether the method is successful or not. When the probe is immobilized, the binding efficiency can be judged by judging the number of immobilized nucleic acid molecules, and for this purpose, an optically detectable label is preferably provided at the 3' -end of the first strand, and fluorescent molecules such as Cy3, cy5, etc. are preferably selected, so that the hybridization efficiency can be easily and intuitively detected.
To capture single-stranded nucleic acid molecules, in one example, double-stranded nucleic acid molecules are melted into single-stranded nucleic acid molecules, for example, by denaturation, after which the probe hybridizes to the single-stranded nucleic acid molecules to form a hybridization complex. The denatured nucleic acid molecules are complementarily bound to the probe in the hybridization solution.
By the above-described capturing method, on the one hand, double-stranded nucleic acid molecules can be captured, and on the other hand, single-stranded nucleic acid molecules can be captured. The double-stranded nucleic acid molecule is captured by the method, on one hand, the target double-stranded molecule can be purified and separated, and on the other hand, a hybridization complex formed after capturing the double-stranded nucleic acid molecule synthesizes a target sequence, such as a probe sequence of a synthesized nucleic acid fragment, in the presence of a strand displacement reaction polymerase. Hybridization complexes formed after capture of single stranded nucleic acid molecules by this method can be used for sequence sequencing to determine specific sequence information for the nucleic acid fragments.
The method for capturing the nucleic acid molecule skillfully utilizes the characteristic that the double-stranded nucleic acid molecule has a single-stranded region, can effectively capture double-stranded nucleic acid sequences, and can capture single-stranded nucleotides. By utilizing the relation between the Tm of the single-chain region and the Tm of the double-chain region, a proper hybridization capture temperature is designed, so that the probability of hybridization between the second chain and the first chain is reduced while nonspecific hybridization is reduced, and the hybridization efficiency of the probe and the first chain is improved.
In a second aspect, the present application provides a hybridization complex obtained by the capture method described above. The hybridization complex has two types, the first type is formed by hybridization of a probe and undenatured nucleic acid molecules, namely double-stranded nucleic acid molecules, the probe and the single-stranded tail end are paired to form the hybridization complex, and the hybridization complex contains three sequences, namely two sequences of the nucleic acid molecules and a probe sequence; the second type is formed by hybridization of a probe with a nucleic acid molecule denatured into single strands, the sequence hybridized with the probe pair contains at least a single-stranded terminal sequence, and the hybridization complex contains two sequences, namely a single-stranded nucleic acid molecule sequence and a probe sequence.
In a third aspect the application provides a kit comprising a nucleic acid molecule as described above. Further, the kit further comprises probes and/or hybridization complexes. The nucleic acid molecule in the kit comprises a first nucleic acid strand and a second nucleic acid strand that are complementary and have at least one single-stranded end located on the first nucleic acid strand, and the probe in the kit is complementary to the single-stranded end sequence of the first nucleic acid strand. The kit can be used in sequence hybridization capture experiments, control experiments of probe sequence hybridization capture experiments and sequencing.
In a fourth aspect the application provides the use of a kit as described above in sequence hybridization capture and/or sequencing. When the kit is used for sequence hybridization capture and/or sequencing, the kit can be used as a control group experiment to control quality of the sequence hybridization capture.
In a fifth aspect the present application provides a sequencing method comprising sequencing a hybridization complex, wherein the hybridization complex is obtained by the method described above. When sequencing is performed, when the hybridization complex is a complex of three sequences, the sequencing is performed using a strand displacement DNA polymerase, such as Phi29 DNA polymerase; when the hybridization complex is a complex of two sequences, sequencing may be performed using a common DNA polymerase for sequencing, such as taq DNA polymerase.
The sixth aspect of the present application provides a method for producing a nucleic acid library, comprising ligating a first nucleic acid fragment and a second nucleic acid fragment to obtain the nucleic acid library, the first nucleic acid fragment and the second nucleic acid fragment being double-stranded DNA, the first nucleic acid fragment being a known sequence, the first nucleic acid fragment comprising a first strand and a second strand which are complementary, the first nucleic acid fragment having a single-stranded end located at the 3' end of the first strand, and 80 ℃ not less than (Tm 1-Tm 2) not less than 10 ℃,90 ℃ not less than Tm1 not less than 50 ℃, tm1 being the dissolution temperature of the first strand, tm2 being the dissolution temperature of the second strand being Tm2.
In one embodiment of the present application, tm1 is 71℃and Tm2 is 45.6 ℃.
It will be appreciated that the above nucleic acid fragments may be linked by chemical bonds using a phosphodiester linkage but are not limited to a phosphodiester linkage, and that when the two are linked by a phosphodiester linkage, the 5' end of the first strand has a phosphate group. The natural nucleotide contains phosphate groups and hydroxyl groups, and is chemically connected by adopting phosphodiester bonds, so that the connection product does not damage the natural structural characteristics of the nucleotide chain.
In one embodiment, the 3' -end of the second strand is free of hydroxyl groups, and the second strand having this feature is used to construct a nucleic acid library, wherein the ligation products in the nucleic acid library have two forms, one form being: the second strand is linked to the second nucleic acid fragment by a non-phosphodiester linkage; another form is: the ligation product does not contain a second strand, and when two strands of the ligation product are used as PCR substrates, the second strand cannot be effectively amplified, and if the ligation product is used as a sequencing substrate, detection of invalid data can be reduced, and the effective detection data amount can be improved.
When the nucleic acid library prepared by the preparation method of the nucleic acid library is used for sequencing, particularly single molecule sequencing, the hybridization efficiency with a probe can be improved, so that the target fragment for effective sequencing is increased. In the embodiment of the application, the first nucleic acid fragment and the blunt end linker are used for respectively constructing a nucleic acid library for probe capture hybridization, the capture hybridization efficiency is compared, the fluorescent signal is detected after the nucleic acid library fragment is captured by the probe to judge the quantity of the captured nucleic acid library, and the capturing efficiency of the nucleic acid library and the probe obtained by the library construction method is more than 2 times of the capturing efficiency of the blunt end linker construction library as seen from the detection result.
The constructed library has various applications such as hybridization of chip sequences, sequencing, etc. When the nucleic acid library and the probe are complementarily combined, the combination condition of the nucleic acid library and the probe, particularly the combination efficiency and the combination position, needs to be intuitively judged, so as to judge whether the operation is successful or not. When the probe is immobilized, the binding efficiency and binding position can be determined by determining the number of immobilized nucleic acid molecules, and for this purpose, a preferred scheme is as follows: the 3' -end of the first strand is provided with an optically detectable label, and fluorescent molecules such as Cy3, cy5 and the like are preferably selected, so that the hybridization efficiency can be simply and intuitively detected.
The manner of ligating the nucleic acid fragments in the nucleic acid library preparation method may be selected from blunt end ligation and/or cohesive end ligation. The nucleic acid fragments with flat ends at both ends can be connected after T/A is added to the ends, so that the connection efficiency can be improved and the connection directionality can be ensured.
When two nucleic acid fragments are connected, the mole amount of a first nucleic acid fragment with a known sequence is more than that of a second nucleic acid fragment, so that the probability of connecting the second nucleic acid fragment with the first nucleic acid fragment is improved, and based on the comparison optimization result of the mole concentration of the two nucleic acid fragments in multiple experiments, the first nucleic acid fragment and the second nucleic acid fragment can be effectively connected when the mixing proportion of the first nucleic acid fragment and the second nucleic acid fragment is in the range of 50-500:1, and when the mixing proportion is in the range of 200:1, more than 90% of the second nucleic acid fragment is connected with the first nucleic acid fragment, and the self-connecting rate (connecting between the second nucleic acid fragments) of the second nucleic acid fragment is greatly reduced.
In one implementation of the present application, the preparation method of the present application specifically includes the following steps: comprises ligating a first nucleic acid fragment and a second nucleic acid fragment to obtain the nucleic acid library, wherein the first nucleic acid fragment and the second nucleic acid fragment are double-stranded DNA, the first nucleic acid fragment is a known sequence, the first nucleic acid fragment comprises a first strand and a second strand which are complementary, the first nucleic acid fragment has a single-stranded end, the single-stranded end is positioned at the 3' end of the first strand, and the temperature of 80 ℃ is more than or equal to (Tm 1-Tm 2) is more than or equal to 10 ℃, the temperature of 90 ℃ is more than or equal to Tm1 is more than or equal to 50 ℃, the Tm1 is the dissolution temperature of the first strand, and the temperature of Tm2 is the dissolution temperature of the second strand is Tm2. Wherein the 3 'end of the second strand is free of hydroxyl groups and the 5' end of the first strand contains phosphate groups, and the first strand is capable of being linked to the second nucleic acid fragment via phosphodiester bonds. The nucleic acid library is constructed using this method, either without a second strand in the ligation product, or the second strand is ligated to a second nucleic acid fragment via a non-phosphodiester linkage. Both forms of ligation product cannot be effectively amplified when both strands of the ligation product are used as PCR substrates, and if the ligation product is used as a sequencing substrate, detection of invalid data can be reduced and the amount of valid detection data can be increased.
The seventh aspect of the present application provides a nucleic acid library, which is a ligation product obtained by the above-described preparation method, wherein the ligation product is a product obtained by directly ligating the nucleic acid fragment provided in the third aspect of the present application with the second nucleic acid fragment or a product obtained by directly ligating the ligation product with n rounds of PCR amplification (n is 1.ltoreq.6, and n is an integer).
In an eighth aspect, the application provides a method of capturing a nucleic acid molecule comprising the steps of:
1) Denaturing the nucleic acid library to form single strands;
2) Hybridizing the single-stranded product of step 1) with a probe to form a hybridization complex.
Wherein the probe comprises an oligonucleotide strand. The oligonucleotide strand at one end of the probe functions to complementarily hybridize to a sequence in a nucleic acid molecule, and it is understood that the other end of the probe may be a nucleotide sequence or other sequence, such as an amino acid sequence, and the sequence at the other end may have other modifications, such as amino modifications, fluorescent modifications, and the like. In one embodiment of the application, the probes are identical in sequence and the 3' -terminal nucleotide contains a hydroxyl group (-OH). Based on the principle of complementary pairing of nucleic acids, it is known that when a probe is paired with a first nucleic acid strand, the probe can be used as a primer when the single-stranded end is located at the 3' -end of the first nucleic acid strand, and the first nucleic acid strand can be used as a template for PCR amplification. Optionally, the oligonucleotide strand is a DNA strand.
The probe may be complementarily paired with all or part of the sequence of the first strand, and preferably one scheme is that all of the sequence of the first strand is complementarily paired with the probe. It will be appreciated that complementary pairing of the first strand with the probe is to ensure preferential hybridization of the first strand with the probe at the chosen annealing temperature, and thus that mismatched sequences are allowed to exist in the paired sequences of all sequences of the first strand with the probe under conditions which meet the above requirements.
For efficient capture of nucleic acid sequences containing the first strand, hybridization temperatures T, tm2-5 ℃ < T.ltoreq.Tm1-5℃are chosen. By selecting an appropriate hybridization temperature, the probability of hybridization of the second strand to the first strand can be reduced, thereby increasing the hybridization efficiency of the probe to the first strand. Meanwhile, the number of nucleic acid fragments (nucleic acid sequences not containing a linker) with the same sequence is far less than that of probes, and the probability of forming double chains by pairing between single chains of the nucleic acid fragments is low at the annealing temperature, so that the influence on the capturing efficiency is negligible due to the number of double chains formed between the nucleic acid fragments. In one embodiment of the application, the annealing temperature is selected to be 55 ℃.
Meanwhile, it is understood that the sequence probe paired with the first nucleic acid strand contains an oligonucleotide strand, and when the oligonucleotide strand is located at one end of the probe, the other end of the probe may be a nucleotide sequence or other sequences, such as an amino acid sequence, and the other end of the probe may have other modifications, such as amino modifications, fluorescent modifications, and the like.
The choice of the oligonucleotide strand in the probe will take into account all factors such as annealing temperature, nucleotide composition, length and internal secondary structure of the oligonucleotide strand, in one embodiment of the application the length of the oligonucleotide strand is 20-80 nt.
Further, the nucleotide at the 3 '-end of the above-mentioned oligonucleotide chain contains a hydroxyl group (-OH) so that the 3' -end of the oligonucleotide chain can be linked to other nucleotides via phosphodiester bonds.
In one embodiment, the probe is immobilized on a solid medium selected from at least one of glass and plastic magnetic beads. The probe is fixed on a solid medium, so that on one hand, the separation and purification of the nucleic acid sequence after the probe captures the nucleic acid sequence are facilitated; alternatively, the capture of the nucleic acid sequence may be followed by other detection or manipulation, such as PCR amplification, sequencing reactions, and the like.
In a ninth aspect the present application provides a sequencing method comprising sequencing a hybridization complex, wherein the hybridization complex is obtained by the method described above. When sequencing is performed, when the hybridization complex is a complex of three sequences, the sequencing is performed using a strand displacement DNA polymerase, such as Phi29 DNA polymerase; when the hybridization complex is a complex of two sequences, sequencing may be performed using a common DNA polymerase for sequencing, such as taq DNA polymerase.
In a tenth aspect the application provides a kit for preparing a nucleic acid library, capturing and/or sequencing, the kit comprising a first nucleic acid fragment, in one example the first nucleic acid fragment is a linker. Further, the kit further comprises a probe capable of capturing the first nucleic acid fragment, wherein the nucleotide at the 3' -end of the probe contains-OH. Based on the principle of complementary pairing of nucleic acids, it is known that when a probe is paired with a first nucleic acid strand, the probe can be used as a primer when the single-stranded end is located at the 3' -end of the first nucleic acid strand, and the first nucleic acid strand can be used as a template for PCR amplification.
It will be appreciated that the oligonucleotide strand at one end of the probe acts to complementarily hybridize to a sequence in a nucleic acid molecule, and that the other end of the probe may be of a nucleotide sequence or of another sequence, such as an amino acid sequence, and that the sequence at the other end may also have other modifications, such as amino modifications, fluorescent modifications, and the like. In one example, the oligonucleotide strand is a DNA strand.
The choice of the oligonucleotide strand in the probe will take into account all factors such as annealing temperature, nucleotide composition, length and internal secondary structure of the oligonucleotide strand, in one embodiment of the application the length of the oligonucleotide strand is 20-80 nt.
In one embodiment, the probe is immobilized on a solid medium selected from at least one of glass, plastic, and magnetic beads.
Drawings
FIG. 1 illustrates the hybridization results of library 1 and library 2 with probes in one embodiment of the present application. The abscissa is for library 1 and library 2, and the ordinate is the number of bright spots (dots) within a particular field of view (FOV, e.g. 110 x 110 μm), expressed as Dot/FOV.
Term interpretation:
as used herein, "nucleic acid", "nucleic acid molecule" and "nucleic acid fragment", including DNA, RNA, RNA-DNA and/or cDNA, double-stranded or single-stranded, are sequences consisting of nucleotides and/or nucleotide analogs (derivatives).
As used herein, a "linker" is a nucleic acid fragment of known sequence.
Examples
The application will now be described with reference to specific embodiments and figures. It should be noted that these examples are illustrative only and are not to be construed as limiting the application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
1. Preparation of the Joint
The nucleotide sequences shown in Table 1 were designed and synthesized.
TABLE 1
Note that: 5' -PHO:5' -terminal phosphorylation modification; 3' Cy3:3' Cy3 modification; 5' -NH 2 C 6 : NH at 5' end 2 Modified, and NH 2 The modified position is the C6 of the nucleotide.
The dissolution temperature Tm of each strand was calculated using on-line software (http:// biotools. Nubic. Northwestern. Edu/oligocalc. Html), SEQ ID NO:1: the dissolution temperature is 45.6 ℃; SEQ ID NO. 2: the dissolution temperature was 71 ℃.
The preparation process of the joint comprises the following steps: the synthesized nucleotide sequences shown in Table 1 were each dissolved to 100 pmol/. Mu.l with 1X linking Buffer (10 mM Tris pH 7.5-8.0, 50mMNaCl,1mM EDTA), and then quantified using Nanodrop 2000. According to the quantitative results, the nucleotide sequences shown in SEQ ID NOS: 1-4 were diluted to 100 pmol/. Mu.l with 1X linking Buffer, respectively.
Mu.l of oligo1.1 and oligo1.2 were mixed in a clean 200. Mu.l PCR tube at a concentration of 100 pmol/. Mu.l, and the mixture was pipetted and homogenized to prepare a linker 1. Similarly, 10. Mu.l of oligo2.1 and 10. Mu.l of oligo2.2 were mixed in another PCR tube at a concentration of 10. Mu.l, 100 pmol/. Mu.l, respectively, to prepare a linker 2. The mixed sequences were subjected to slow annealing in a PCR instrument to prepare linker 1 and linker 2, and the linker preparation set conditions are shown in table 2:
TABLE 2
The prepared joints 1 and 2 were measured with a Qubit 2.0 meter, qubit TM dsDNA BR Assay Kit, quantification was performed.
2. Library construction
Repair of the DNA fragment and ligation were performed using a kit of norpran (VAHTS Universal DNA library prep kit for Illumina Vazyme cat, cat No. ND 606), wherein the molar ratio of DNA fragment to ligation was 1:200, i.e. DNA: linker = 1:200. Library 1 was constructed using linker 1 and library 2 was constructed using linker 2. Library 1 and library 2 were constructed under identical conditions with only the linker used. The specific operation steps are as follows:
step one: end repair
The ends of DNA fragmented with a length of 100-500bp (main band of 200 bp) were filled in, and phosphorylated at the 5 'end and dA tail at the 3' end.
The end fill-up reaction system was configured in a sterilized PCR tube with the specific composition shown in table 3:
TABLE 3 Table 3
| Component (A) | Volume of |
| DNA (38 nM concentration) | 10μl |
| End Prep Mix 4 | 15μl |
| ddH2O | To 65μl |
And (3) uniformly mixing the end-fill reaction system, and collecting the reaction liquid to the bottom of the tube by short centrifugation.
The end repair reaction conditions are shown in table 4:
TABLE 4 Table 4
| Temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 20℃ | 15min |
| 65℃ | 15min |
| 4℃ | Hold |
And obtaining a terminal repair product after the reaction is finished.
Step two: joint connection
And (3) connecting the product subjected to the end repair in the step one with a joint.
(1) In the first step, a linker ligation reaction system was prepared in the PCR tube with the repaired end, and the linker ligation reaction system is shown in Table 5:
TABLE 5
| Component (A) | Volume of |
| End repair products | 65μl |
| Rapid Ligation buffer 2 | 25μl |
| Rapid DNA ligase | 5μl |
| Joint 1 or joint 2 (15 uM concentration) | 5μl |
| Totals to | 100μl |
The mixture was gently stirred with a pipette and centrifuged briefly to collect the reaction solution to the bottom of the PCR tube.
(2) Placing the PCR tube of the joint connection reaction system prepared in the step (1) into a PCR instrument for reaction, wherein the joint connection reaction conditions are set according to the conditions shown in Table 6:
TABLE 6
| Temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 20℃ | 15min |
| 4℃ | Hold |
And obtaining a joint connection product after the reaction is finished.
Step three: purification
The linker ligation product of step two (2) was purified using VAHTS DNA Clean Beads, the purification steps were as follows:
a) After the magnetic beads were equilibrated to room temperature, vortex shaking was performed VAHTS DNA Clean Beads.
b) Pipette 60. Mu. l VAHTS DNA Clean Beads to 100. Mu.l of the adaptor-ligated product, vortex or gently blow 10 times with a pipette and mix well.
c) Incubate at room temperature for 5min.
d) The PCR tube was briefly centrifuged and placed in a magnetic rack to separate the beads from the liquid, after which the solution was clarified (about 5 min) and the supernatant carefully removed.
e) The PCR tube was kept always in a magnetic rack, the beads were rinsed with 200. Mu.l of freshly prepared 80% ethanol, incubated for 30sec at room temperature, and the supernatant carefully removed.
f) Step 5 was repeated for a total of two rinses.
g) The PCR tube is kept to be always placed in the magnetic frame, and the magnetic beads are air-dried after being uncapped for 5-10min until no ethanol remains.
h) The PCR tube is taken out from the magnetic frame for elution:
add 22.5. Mu.l of eluent (10 mM Tris-HCl, pH 8.0-pH 8.5), elute, vortex or gently blow with a pipette to mix well, place at room temperature for 2min, briefly centrifuge the PCR tube and place in a magnetic rack for standing, after the solution is clear (about 5 min), carefully remove 20. Mu.l of supernatant into the fresh EP tube, and do not touch the magnetic beads.
The purified library was quantified using a Qubit 2.0 quantifier, qubitTM dsDNA BR Assay Kit, with linker 1 for library 1 and linker 2 for library 2.
3. Hybrid capture
The probes (SEQ ID NO: 4) were immobilized on a chip using the method disclosed in the specification of published patent application CN201510501968.7, and library 1 and library 2 prepared were diluted with 3 XSSC hybridization solution and then hybridized with the probes immobilized on the chip. The number of hybridization of the linker sequence to the probe was then determined based on the Cy3 signal.
The procedure for hybridization of library chips was as follows:
(1) Chip selection: the base glass of the chip used was an epoxy-modified glass chip of the company SCHOTT, and the probe (sequence: TTTTTTTTTTTCCTTGATACCTGCGACCATCCAGTTCCACTCAGATGTGTATAAGAGACAG) was immobilized by reacting an amino group on the probe with an epoxy group on the surface of the chip, for example, as disclosed in published patent application No. CN201811191589.2, and the density of the immobilized probe in a 110X 110. Mu.M region was about 18000Dot/FOV, i.e., 18000 bright spots in a 110X 110. Mu.M field of view.
(2) Hybridization solution preparation: the hybridization solution preparation system is shown in Table 7, and the buffer used is 20 XSSC buffer (sigma, # S6639-1L) at a final concentration of 3 XSSC, the library at a final concentration of 1nM, and the total volume of 40. Mu.L. The prepared hybridization solution is denatured for 2min at 95 ℃ and rapidly cooled on ice.
TABLE 7
| 20 XSSC buffer | 6uL |
| Library 1 or library 2 | Final concentration of 1nM |
| Nucleic acid-removing water | Make up to 40 mu L |
(3) And (3) rapidly loading the denatured hybridization solution onto a chip, and placing the chip at 55 ℃ for 30min to hybridize the library with probes on the surface of the chip.
(4) The chip was washed with 3 XSSC, 1 XSSC, 0.1 XSSC in this order.
(5) The chip was then placed on a sequencer and photographed at 532 wavelength, counting the number of points in each field of view.
4. Experimental results
The experimental results are shown in FIG. 1, in which the abscissa is for library 1 and library 2, and the ordinate is Dot/FOV (the number of bright spots in the observation area is 110X 110 μm). Library 1 hybridized with the probe at the same hybridization concentration and the same field of view (in the region of 110X 110. Mu.M), the hybridization density (average 14400 light spots) of library 1 was much higher than that of library 2 (average 7000 light spots), and the difference between the hybridization densities of library 1 and probe was found to be higher than that of library 2.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.
Sequence listing
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Claims (14)
1. A method of sequencing comprising:
preparing a nucleic acid library comprising ligating a linker and a second nucleic acid fragment, obtaining a nucleic acid library, said linker comprising a first strand and a second strand that are complementary, said nucleic acid library comprising a first nucleic acid strand and a second nucleic acid strand that are complementary, said nucleic acid library having at least one single-stranded end, at least one of said single-stranded ends being located on said first nucleic acid strand, said single-stranded end being located on said first strand,
hybridization, comprising hybridizing at least a portion of a first strand of the nucleic acid library with a probe at a hybridization temperature T to obtain a hybridization complex, wherein Tm2-5 ℃ is less than or equal to T is less than or equal to Tm1-5 ℃,80 ℃ is less than or equal to (Tm 1-Tm 2) is less than or equal to 10 ℃, and 90 ℃ is less than or equal to Tm1 is less than or equal to 50 ℃, tm1 is the dissolution temperature of the first strand, tm2 is the dissolution temperature of the second strand,
sequencing the hybridization complex by using DNA polymerase with a strand displacement function when the hybridization complex is formed by hybridization of a probe and undenatured double-stranded nucleic acid molecules in the nucleic acid library.
2. The method of claim 1, wherein the single stranded end is located at the 3' end of the first nucleic acid strand.
3. The method of claim 1, wherein the probe is single stranded oligonucleotide.
4. A method according to claim 3, wherein the probe has a length of 20 to 80nt.
5. A method according to claim 3, wherein the nucleotide at the 3' end of the probe contains a hydroxyl group.
6. The method of claim 3, wherein the probe is immobilized on a solid medium selected from at least one of glass, plastic, and magnetic beads.
7. The method of claim 6, wherein the adaptor and the second nucleic acid fragment are ligated by blunt end ligation and/or cohesive end to obtain the nucleic acid library.
8. The method of claim 7, wherein the 5' end of the first strand has a phosphate group.
9. The method of claim 7, wherein the 3' end of the second strand is free of hydroxyl groups.
10. The method of claim 9, wherein all sequences of the first strand are complementarily paired to probes.
11. The method of claim 1, wherein Tm1 is 71 ℃, and Tm2 is 45.6 ℃.
12. The method of claim 1, wherein the 3' end of the first strand carries an optically detectable label.
13. The method of claim 12, wherein the optically detectable label is a fluorescent molecule.
14. Use of the method of any one of claims 1-13 for sequence hybridization capture and/or sequencing.
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| CN202311464676.1A CN117535379A (en) | 2019-08-09 | 2019-08-09 | Method for capturing nucleic acid molecules and application thereof |
| PCT/CN2020/107695 WO2021027706A1 (en) | 2019-08-09 | 2020-08-07 | Method for capturing nucleic acid molecule, preparation method for nucleic acid library, and a sequencing method. |
| EP20853500.5A EP4012029B1 (en) | 2019-08-09 | 2020-08-07 | Method for capturing nucleic acid molecule, preparation method for nucleic acid library, and a sequencing method |
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