WO2021052310A1 - 一种dna文库构建方法 - Google Patents

一种dna文库构建方法 Download PDF

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WO2021052310A1
WO2021052310A1 PCT/CN2020/115275 CN2020115275W WO2021052310A1 WO 2021052310 A1 WO2021052310 A1 WO 2021052310A1 CN 2020115275 W CN2020115275 W CN 2020115275W WO 2021052310 A1 WO2021052310 A1 WO 2021052310A1
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linker
dna
mixture
ligase
sticky ends
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PCT/CN2020/115275
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English (en)
French (fr)
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李剑力
张静澜
张治位
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北京博昊云天科技有限公司
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Publication of WO2021052310A1 publication Critical patent/WO2021052310A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the present invention provides a method for constructing a DNA library, a method for sequencing one or more target nucleic acids using the library, a composition, and a kit.
  • Nucleic acid sequences encode information necessary for biological function and reproduction. Therefore, sequencing the accounting sequence is a very important basic technology for research on living organisms and for applications such as disease diagnosis and drug development.
  • sequencing methods are usually used to diagnose and treat various diseases, including cancer, infectious diseases, autoimmune diseases, genetic diseases, and so on.
  • higher throughput and/or lower cost nucleic acid sequencing technologies are required, and on the other hand, it is also necessary to be able to sequence even smaller amounts of nucleic acids. Sequencing of trace nucleic acids is particularly important in certain situations, such as the detection of fetal-related nucleic acid sequences by sequencing free nucleic acids in maternal peripheral blood, and the diagnosis of cancer by sequencing free nucleic acids in patients' peripheral blood.
  • the current next generation sequencing is based on the construction of a sequencing library.
  • the library construction process usually includes: (1) Ultrasonic interruption of unfragmented genomic DNA, or enzyme fragmentation treatment (such as using plasma free DNA or other fragmented DNA, this step can be omitted); (2) DNA The fragments are repaired with end gaps; (3) Phosphorylate the 5'end of the DNA fragment after end repair, and add a single base A (adenine) to the 3'end to obtain a single base sticky end; (4) DNA fragments with sticky ends A are connected to adapters designed according to different sequencing platforms (the adapters can carry sample tags) to obtain the ligation products; (5) If it is to sequence the targeted enriched region (such as whole exome sequencing or NGS panel sequencing), PCR is performed on the ligated products through the universal primers of each sequencing platform on the adapter to obtain a sufficient amount of amplified DNA library (sample tags can also be introduced at this step); (6) After amplification Use probe capture method or targeted primer method to enrich the target region to prepare sequencing library
  • the design of the NGS library construction method will directly affect the conversion rate of the starting DNA into the library by ligation with the adapter, and further affect the yield of the final sequencing library and the DNA complexity of the starting DNA molecules that can be analyzed.
  • the common problems of traditional methods in library construction include: DNA blunt ends or end-repaired DNA fragments with base A added to the 3'end of the single-base sticky end to obtain low ligation efficiency, and incomplete phosphorylation at the 5'end prevents incomplete phosphorylation.
  • the non-specific addition of base A (incorrect addition of G, C, T) at the 3'end hinders the effective AT connection between the linker and the DNA, and the self-ligation between the linker and the target DNA prevents the linker and the DNA from being connected.
  • the 5'end and the 3'end require a higher amount of starting DNA. If the starting DNA is insufficient, PCR amplification is required to increase the library yield to facilitate downstream samples in the process deal with. However, PCR amplification will introduce repetitive sequences and errors and reduce the quality of sequencing. Take cell free DNA (cfDNA) as an example. 1ng of cfDNA contains about 330 genome copies. If 10ng of starting DNA is used, the final theoretical maximum sequencing depth should be 3300. However, using traditional NGS library construction methods, Part of the starting DNA could not complete the conversion rate of the library. Finally, the effective sequencing depth in clinical applications is generally only a small part of the theoretical sequencing depth.
  • the present invention develops a new DNA sequencing or genotyping library construction method, which improves the diversity of linkers, so that it can effectively connect with natural DNA with different ends and DNA fragmented by mechanical or enzymatic methods.
  • the present invention provides a DNA library construction method, wherein the method includes the following steps: (1) dephosphorylating the 5'end of the DNA fragment; (2) providing a linker mixture, the linker mixture comprising Blunt end adaptors and adaptors with sticky ends of different lengths; (3) Connect the 5'end of the adaptor in the adaptor mixture to the 3'end of the dephosphorylated DNA fragment; (4) Use the nick translation method The 3'end of the adaptor in the adaptor mix is connected to the 5'end of the DNA fragment.
  • the DNA library constructed by the invention can be used for sequencing, genotyping and other purposes.
  • the DNA library construction method provided by the present invention is not restricted by any specific sequencing or genotyping platform, and can be widely applied to different DNA analysis platforms.
  • the library construction method provided by the present invention is suitable for constructing libraries for DNA fragments of different lengths.
  • the length of the DNA fragment used in the method of the present invention is, for example, less than 10000bp, 1000bp, less than 900bp, less than 800bp, less than 700bp, less than 600bp, less than 500bp, less than 400bp, less than 300bp, less than 200bp, less than 150bp, less than 100bp , Less than 90bp, less than 80bp, less than 70bp, less than 60bp, less than 55bp, less than 50bp, less than 45bp, less than 40bp or less than 35bp.
  • the lengths of the sticky ends of the linkers with sticky ends of different lengths are 1-10 bp, 1-9 bp, 1-8 bp, 1-7 bp, 1-6 bp, 1-5 bp, 1-4 bp , 1-3bp, 1-2bp or 1bp.
  • the sticky end is a 5'sticky end and/or a 3'sticky end.
  • the sticky end is a random nucleotide sequence, such as a random deoxynucleotide sequence (that is, each position of the sticky end can be A, T, C, G or modified A, One of T, C, G), thereby forming a random linker mixture.
  • the linker with sticky ends of different lengths includes a universal sequence and a random sequence, wherein the random sequence part is the sticky end.
  • at least one linker of the linker mixture may comprise a unique molecular barcode and/or a sample label, wherein the single molecular barcode and/or sample label may be located in the double-stranded part of the linker, It can also exist only as a single strand. When it exists as a single strand, it is located at the opposite end of the DNA fragment (that is, the end that does not participate in the link with the target DNA fragment). For linkers with sticky ends, the single-molecule label and/or sample label existing as a single strand is located at the other end opposite to the sticky end.
  • the other end of at least one of the adaptors in the adaptor mixture opposite to the end connected to the DNA fragment is closed by a circular sequence.
  • the specific shape of the adaptor is shown in Figure 1B. .
  • the total length of at least one linker in the linker mixture (that is, the sum of the double-stranded part and the single-stranded part, the single-stranded part may only be present at one end, or may be present at both ends) is 21-53bp , 21-54bp, 21-55bp, 21-56bp, 21-57bp, 22-54bp, 23-55bp, 24-56bp, 25-57bp, 21-87bp, 22-88bp, 23-89bp, 24-90bp, 25 -91bp, 26-92bp, 27-93bp, 28-94bp, 29-95bp, or 30-96bp.
  • the length of the double-stranded portion is no shorter than 5 bp, no shorter than 10 bp, no shorter than 15 bp, or no shorter than 20 bp.
  • at least one of the linkers in the linker mixture has SpC3 (C3 spacer group) or modified nucleotides at the 3'end (ie, the 3'end of the sense strand) that is connected to DNA, and
  • the modified nucleotide may be dideoxythymidine, dideoxycytidine, dideoxyguanosine, or dideoxyadenosine.
  • the nucleotide library construction method further includes the step of fragmenting the DNA before dephosphorylating the 5'end of the DNA fragment.
  • the fragmentation method includes ultrasonic interruption, enzyme fragmentation treatment and the like.
  • the present invention does not limit the method of fragmenting DNA, as long as the method can make larger fragments of DNA form DNA fragments of a suitable size for the method of the present invention, all methods can be used in the method of the present invention.
  • the DNA library construction method further includes step (5) after step (4): amplifying the DNA fragments connected to the adaptor.
  • amplification primers can be designed for the universal sequence, so as to perform amplification by methods such as PCR.
  • the present invention does not limit the DNA amplification method, and any amplification method known in the art can be applied to the present invention. Examples of amplification techniques that can be used include, but are not limited to, PCR, quantitative PCR, quantitative fluorescent PCR (QF-PCR), multiple fluorescent PCR (MF-PCR), real-time PCR (RT-PCR), in situ rolling circle amplification (RCA), ligase chain reaction (LCR), etc.
  • the sample tag is added in the amplification step by adding the sample tag to the amplification primer.
  • step (3) the 5'end of the linker in the linker mixture is ligated to the 3'end of the dephosphorylated DNA fragment by using a ligase.
  • the present invention does not intend to limit the ligase used, as long as any ligase that can connect the 5'end of the linker to the 3'end of the DNA fragment in the linker mixture can be used in the method of the present invention.
  • Commonly used ligases include but are not limited to: NEB Blunt TA ligase mixture, T7 ligase, T4 ligase, and mixtures of two or more of them.
  • step (4) the 3'end of the linker in the linker mixture is connected to the 5'end of the DNA fragment by any nick translation method known to those skilled in the art. Since the 5'end of the fragmented DNA fragment is dephosphorylated, although the adaptor can bind to the negative strand of the target DNA fragment through complementary sequences, its 3'end cannot be connected to the 5'end of the target DNA fragment in step (3) To form a connection, it is necessary to connect through the incision translation method.
  • the nick translation method in this step uses Taq DNA polymerase and E Coli ligase in combination.
  • the reaction temperature of the nick translation method in this step is 30.0 to 50.0°C, 35.0 to 45.0°C, 36.0 to 44.0°C, 37.0 to 43.0°C, 38.0 to 42.0°C, 39.0 to 41.0°C, or 40°C.
  • a step of adding a 5'end linker single-stranded nucleotide to the reactant is further included before the reaction in step (4).
  • the 5'end linker single strand refers to a single strand connected to the 5'end of the target DNA.
  • the present invention omits the experimental steps of repairing the target DNA ends, 5'end phosphorylation, and 3'end addition A.
  • the use of different lengths of sticky end hybrid joints significantly improves the connection efficiency.
  • dephosphorylating the 5'end of the DNA fragment and modifying the 3'end of the adaptor the self-ligation of the DNA fragment and the adaptor itself is reduced, thereby greatly improving the library construction conversion rate.
  • the present invention provides a kit for constructing a DNA library, the kit including a linker mixture including a blunt end linker and a linker with sticky ends of different lengths.
  • the lengths of the sticky ends of the linkers with sticky ends of different lengths are 1-10 bp, 1-9 bp, 1-8 bp, 1-7 bp, 1-6 bp, 1-5 bp, 1-4 bp , 1-3bp, 1-2bp or 1bp.
  • the sticky end is a 5'sticky end and/or a 3'sticky end.
  • the sticky end is a random nucleotide sequence, such as a random deoxynucleotide sequence (that is, each position of the sticky end can be A, T, C, G, or a modified A , T, C, G) to form a random linker mixture.
  • the linker with sticky ends of different lengths includes a universal sequence and a random sequence, wherein the random sequence part is the sticky end.
  • at least one linker of the linker mixture may comprise a unique molecular barcode and/or a sample label, wherein the single molecular barcode and/or sample label may be located in the double-stranded part of the linker, It can also exist only as a single strand.
  • the single-molecule label and/or sample label existing as a single strand is located at the other end opposite to the sticky end.
  • the other end of at least one of the adaptors in the adaptor mixture opposite to the end connected to the DNA fragment is closed by a circular sequence.
  • the specific shape of the adaptor is shown in Figure 1B. .
  • the total length of at least one linker in the linker mixture (that is, the sum of the double-stranded part and the single-stranded part, the single-stranded part may only be present at one end, or may be present at both ends) is 21- 53bp, 21-54bp, 21-55bp, 21-56bp, 21-57bp, 22-54bp, 23-55bp, 24-56bp, 25-57bp, 21-87bp, 22-88bp, 23-89bp, 24-90bp, 25-91bp, 26-92bp, 27-93bp, 28-94bp, 29-95bp, or 30-96bp.
  • the length of the double-stranded portion is no shorter than 5 bp, no shorter than 10 bp, no shorter than 15 bp, or no shorter than 20 bp.
  • at least one of the linkers in the linker mixture has a SpC3 blocker or a modified nucleotide at the 3'end (ie, the 3'end of the sense strand) connected to the DNA, and the modified core
  • the glycidyl acid may be dideoxythymidine, dideoxycytidine, dideoxyguanosine, or dideoxyadenosine.
  • the kit for constructing a DNA library provided by the present invention further includes a ligase and/or other reagents.
  • the ligase includes but is not limited to: NEB Blunt TA ligase mixture, T7 ligase, T4 ligase, and mixtures of two or more of them.
  • the kit for constructing a DNA library provided by the present invention further contains enzymes and/or other reagents for the nick translation method.
  • the enzymes used in the nick translation method are Taq DNA polymerase and E Coli ligase.
  • the kit for constructing a DNA library provided by the present invention further comprises enzymes and/or other reagents for DNA amplification.
  • library refers to a plurality of nucleic acids, for example, a plurality of different nucleic acids.
  • DNA library refers to a plurality of DNAs, such as a plurality of different DNAs.
  • DNA can be any polymer containing deoxyribonucleotides, including but not limited to modified or unmodified DNA.
  • genomic DNA can be extracted from a sample, and free DNA, such as plasma free DNA (cfDNA), can also be extracted.
  • the DNA sample may be derived from at least one of mammals, plants, and microorganisms.
  • the genomic DNA may be human whole blood genomic DNA, preferably peripheral blood mononuclear cell genomic DNA. In other embodiments, the DNA is free DNA in human peripheral blood.
  • linker refers to any DNA fragment that can be connected to a target DNA fragment.
  • the joints used in the present invention include blunt end joints and sticky end joints.
  • a “sticky end linker” refers to a linker that is expected to have a single-stranded protruding end to be connected to a DNA fragment.
  • the sticky end ie, the protruding single strand
  • the sticky end has a length of 1-10 bp, 1-9 bp, 1-8 bp, 1-7 bp, 1-6 bp, 1-5 bp, 1-4 bp, 1-3 bp or 1-2 bp.
  • the sticky end may be a 5'sticky end (i.e., a protruding single strand is the 5'end) or a 5'sticky end (i.e., a protruding single strand is the 3'end).
  • the sticky end (ie, the single-stranded protruding) in the sticky end linker is a random sequence, that is, a sequence composed of A, T, C, or G (or modified A, T, C, or G) randomly .
  • the sticky end when the sticky end is 1bp, it can be A, T, C, or G; when the sticky end is 2bp, it can be AA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, GG; and so on.
  • the sticky end of the random sequence can hybridize with the target DNA fragment with the corresponding complementary sticky end sequence, so that the 5'end of the linker DNA and the 3'end of the target DNA can be connected by ligase.
  • “Blunt end adaptor” is the opposite of "sticky end adaptor", which refers to a linker that does not have a single-stranded protruding end that is expected to be connected to a DNA fragment.
  • the two DNA single strands of the linker used in the present invention are referred to as “5' end linker single strand” (or “sense strand”) and “3' end, respectively.
  • Linker single strand (or “antisense strand”).
  • “5' end linker single strand” refers to the single strand connected to the 5'end of the target DNA fragment
  • “3' end linker single strand” (or “antisense strand”) refers to A single strand connected to the 3'end of the target DNA fragment.
  • the 5'end linker single strand (sense strand) cannot be ligated with the 5'end of the target DNA fragment by ligase.
  • the 5'end refers to the 5'end connected to the DNA, that is, the 5'end of the "3' end linker single strand” (antisense strand);
  • the 3'end refers to the 3'end involved in the connection with DNA, that is, the 3'end of the "5' end linker single strand” (sense strand).
  • the linker of the invention comprises a "universal sequence".
  • "Universal sequence” refers to a sequence contained in two or more linkers in a linker mixture. Unlike random sequences, universal sequences are generally known sequences.
  • the universal sequence can be a single molecule label and/or a sample label.
  • all fragments containing the universal sequence can be subjected to the same or similar operations and/or treatments, for example, amplification, identification, and sequencing, using similar methods or techniques (for example, using the same primers or probes) , Separation, etc.
  • the universal sequence provides a common primer binding site for a collection of different target DNAs.
  • the present invention provides a DNA library construction method, wherein the method includes the following steps: (1) dephosphorylating the 5'end of the DNA fragment; (2) providing a linker mixture, the linker mixture Including blunt-end adapters and adapters with sticky ends of different lengths; (3) connecting the 5'end of the adapter in the adapter mixture to the 3'end of the dephosphorylated DNA fragment; (4) making the adapter mixture by the nick translation method The 3'end of the middle linker is connected to the 5'end of the DNA fragment.
  • the present invention omits the repair and complementation of target DNA, and omits the experimental steps of phosphorylation at the 5'end and addition of A at the 3'end, thereby making the experiment easier to operate and quality control.
  • Hybrid joints with sticky ends greatly improve the connection efficiency, and 5'end dephosphorylation solves the problem of self-ligation of the target.
  • the use of adapter mixtures can improve the transformation efficiency of DNA library construction.
  • the present invention also provides a kit for constructing a DNA library.
  • the kit includes a linker mixture, and the linker mixture includes a blunt end linker and a linker with sticky ends of different lengths.
  • the kit for preparing a DNA library provided by the present invention further includes a ligase, an enzyme for the nick translation method, and/or other reagents.
  • the kit provided by the present invention can be used in the DNA library construction method of the present invention.
  • the method provided by the present invention is not limited to specific applications, but is widely used in research, clinical, medicine and other biology, biochemistry and molecular biology. Exemplary uses include genetics and genomics, such as identifying mutations and/or alleles.
  • the method of the present invention can be used to detect whether there is an abnormality in the gene of the fetus, and the like.
  • the method provided by the present invention can also be used in the field of infectious diseases, such as identifying infectious sources such as viruses, bacteria, and fungi, determining virus types and families, and identifying mutations and/or alleles.
  • the method can be used in oncology and related fields of oncology.
  • a specific and non-limiting illustrative example in the field of oncology is the identification of genetic and/or genomic mutations, alleles, etc. associated with cancer.
  • the technology can be used for cancer screening, cancer diagnosis, cancer prognosis, and selection of cancer treatment methods.
  • Figure 1A shows a schematic diagram of the library construction method of the present invention.
  • the steps from left to right are: provide DNA fragment; dephosphorylate the 5'end of the DNA fragment; connect the 3'end of the dephosphorylated DNA fragment to the 5'end of the adaptor; and connect the 5'end of the DNA fragment to the 3'end of the adaptor.
  • the ends are connected by the nick translation method; amplified by PCR and the sample label is introduced.
  • Fig. 1B is a schematic diagram of the shape of a part of the joint with sticky ends of the present invention.
  • Figure 2 shows the effect of different concentrations of ligase on ligation efficiency.
  • Figure 3 shows the effect of different lengths of cohesive ends on the connection efficiency.
  • Figure 4 shows the effect of the modification of the 3'end of the adaptor DNA on the ligation efficiency.
  • Figure 5 shows the effect of dideoxythymidine end modification on the self-ligation of the linker.
  • Figure 6 shows the ligation efficiency of the modified linker with the 3'end of dideoxythymidine.
  • Figure 7 shows the effect of different ligases on the ligation efficiency of the 3'end viscous hybrid linker.
  • Figure 8 shows the effect of different ligases on the ligation efficiency of the 5'end viscous hybrid linker.
  • Figure 9 shows that the 3'end of the adaptor and the 5'end of the target DNA can be connected by the nick translation method.
  • Figure 10 shows the effect of temperature on the nick translation method to ligate the 3'end of the adaptor and the 5'end of the target DNA fragment.
  • the number shown in the figure is temperature (°C).
  • Figure 11 shows the effect of thermostable ligase on the nick translation method to ligate the 3'end of the adaptor and the 5'end of the target DNA fragment.
  • the number shown in the figure is temperature (°C).
  • Figure 12 shows the effect of pretreatment of genomic DNA on the ligation of cohesive linkers.
  • Figure 13 shows the result of using the method of the present invention to construct a library of trace free DNA.
  • ideoxyU means deoxyglucoside
  • N means degenerate base (for example, the position can be A, T, C or G)
  • * means phosphorothioate bond
  • Phos means terminal phosphorylation modification
  • SpC3 means C3 spacer group
  • Biosg means biotin label
  • 6-FAM means 6-carboxyfluorescein
  • ddT means dideoxythymidine
  • DdN means dideoxythymidine, dideoxycytidine, dideoxyguanosine, or dideoxyadenosine.
  • Example 1 The influence of different concentrations of ligase on ligation efficiency
  • Experimental materials oligonucleotide chain h3c0, h3control, subm1 and subnofam, the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB T4 ligase, NEB Blunt TA ligase mixture, DNAse/RNAse-free water, and Gelred.
  • oligonucleotide chains h3c0, h3control, subm1 and subnofam.
  • the oligonucleotide chain is renatured to generate linkers and target DNA respectively.
  • the following steps are used for renaturation: Centrifuge each oligonucleotide pellet briefly and dissolve it in double buffer (100mM potassium acetate; 30mM HEPES, pH 7.5). The two oligonucleotides are polymerized together in equimolar amounts. Heat the mixed oligonucleotides to 94°C for 2 minutes and gradually cool down at room temperature.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer.
  • the reaction buffer includes T4 DNA ligase buffer, 10 pmole DNA fragments, 50 pmole adapters, 400, 800, or 1200 units of T4 DNA ligase or 1800 units of NEB Blunt TA ligase and water .
  • the reaction mixture was incubated at 25°C for 15 minutes, the final product was diluted 5 times and electrophoresed on a 50 volt 5% TBE gel for 105 minutes, and quantified using Agilent Bioanalyzer 2100.
  • Example 2 The effect of different lengths of joint sticky ends on connection efficiency
  • the single-stranded oligonucleotide chains of the synthetic linker are h3c0, h3s0, h3s1, h3s2, h3s3, and the single-stranded oligonucleotide chains of the synthesized target DNA fragment are subnofam, sub0, sub5p1, sub5p2, sub5p3 and submn1, the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, DNAse/RNAse-free water, and Gelred.
  • connection efficiency (84%) of sticky joints with protruding length greater than 1bp is significantly higher than that of blunt-end joints (5%).
  • the linking efficiency of the linker is less than 11% (Figure 3).
  • Example 3 The effect of the modification of the 3'end of the adaptor DNA on the ligation efficiency
  • the single-stranded oligonucleotides of synthetic linker are h3c0ddT, h3c0, h3s0, h3s1, h3s2, h3s3, h3control, and the single-stranded oligonucleotides of synthetic target DNA are subnofam, sub0, sub5p1, sub5p2 , Sub5p3, the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, DNAse/RNAse-free water, and Gelred.
  • the modification of the 3'end of the linker has a significant effect on the ligation efficiency.
  • the 3'end dideoxythymidine has a significantly higher ligation efficiency than the 3'end SpC3 blocker.
  • the linking efficiency of the blunt end linker is similar to that of the 3bp sticky end.
  • a high concentration of ligase such as NEB Blunt TA ligase mixture is used. Since the broken DNA ends can be blunt ends or sticky ends, the linker mixture of the present invention can be efficiently connected to various fragmented target DNAs, and the concentration of ligase can be reduced to reduce experimental costs.
  • the single-stranded oligonucleotides of the synthetic linker are bioh3cm1, h3c0ddT, h3s0s, h3s1s, h3s2s, h3s3s, and the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, DNAse/RNAse-free water, and Gelred.
  • the single-stranded oligonucleotides for synthetic linker are bioh3cm1, h3c0ddT, h3s0s, h3s1s, h3s2s, h3s3s, h3s4s, and the single-stranded oligonucleotides for synthesis of the corresponding target DNA fragments are subfam, sub0spc3, sub5p1, sub5p2, sub5p3, sub5p4.
  • the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, DNAse/RNAse-free water, and Gelred.
  • the negative strand of the target DNA fragment (the strand connected to the linker at the 3'end) has a fluorescent protein FAM at the 5'end, and an overhang of 0, 1, 2, 3, and 4 bases at the 3'end.
  • the mixture was incubated at 25°C for 15 minutes.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour. Photograph the fluorescently labeled ligation product on the DR22A DR Translluminator. Then the gel was post-stained in 3X Gelred solution to detect the size of the ligation product.
  • Example 6 The effect of different ligases on the ligation efficiency of the 3'end viscous hybrid linker
  • the single-stranded oligonucleotides for the synthetic linker are h3c0ddT, h3s0S, h3s1s, h3s2s, h3s3s, h3s4s, and the single-stranded oligonucleotides for the synthesis of the corresponding target DNA fragment are subfam, sub0spc3, sub5p1, sub5p2, sub5p3, sub5p4, The oligonucleotide sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, T7 ligase, T4 ligase, DNAse/RNAse-free water and Gelred.
  • the operation steps were to form the linker and artificially synthesize the target DNA fragment.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer.
  • the reaction buffer included different ligases (see Figures 7A and B for ligase and its dosage.
  • the number after each ligase is the amount of ligase, in microliters) and Ligase buffer mixture, 50 pmole linker mixture, 5 pmole target DNA mixture and water.
  • the linker mixture is formed by mixing linkers with different 3'overhangs in an equimolar ratio
  • the target DNA mixture is formed by mixing target DNAs with different 5'overhangs in an equimolar ratio.
  • the ligase tested in this experiment included a combination of NEB Blunt TA ligase, T7 ligase, T4 ligase, T7 ligase, and T4 ligase.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour. Then the gel was photographed on the DR22A DR Translluminator of the fluorescently labeled ligation product. Then the gel was post-stained in 3X Gelred solution to detect the size of the ligation product.
  • Example 7 The influence of different ligases on the ligation efficiency of the 5'end viscous hybrid linker
  • the single-stranded oligonucleotides of synthetic linker are h3c1, h3c2, h3c3, h3c4, h3s0s, and the single-stranded oligonucleotides of synthetic corresponding DNA fragments are sub0spc3, sub3p1, sub3p2, sub3p3, and sub3p4.
  • the sequence information is shown in Table 1 above.
  • Other experimental materials include NEB Blunt TA ligase mixture, T7 ligase, T4 ligase, DNAse/RNAse-free water, and Gelred.
  • the operation steps were to form the linker and artificially synthesize the target DNA fragment.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer, which included 5 ⁇ L different ligase and ligase buffer mixtures, 50 pmole linker mixture, 5 pmole target DNA mixture and water.
  • the linker mixture is formed by mixing linkers with different 5'overhangs in an equal molar ratio
  • the target DNA mixture is formed by mixing target DNAs with different 3'overhangs in an equal molar ratio.
  • the ligase tested in this experiment included a combination of NEB Blunt TA ligase, T7 ligase, T4 ligase, T7 ligase, and T4 ligase.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour, and then the gel was post-stained in a 3X Gelred solution to detect whether there were ligation products and the size of the ligation products.
  • Example 8 Connecting the 3'end of the adaptor and the 5'end of the target DNA by the nick translation method
  • the single-stranded oligonucleotides for the synthesis of linkers are h5u and h5un, and the single-stranded oligonucleotides for the synthesis of corresponding DNA fragments are sub0fam and h3sub.
  • the sequence information is shown in Table 1 above.
  • Other experimental materials include dNTPs, Taq DNA polymerase, E Coli DNA ligase, 1X E Coli ligase buffer, water, Gelred.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer.
  • the reaction buffer included 40 pmole linker, 20 pmole target DNA, water, dNT Ps, Taq DNA polymerase, E Coli ligase and E Coli ligase buffer.
  • the mixture was incubated at 40°C for 30 minutes.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour.
  • the gel was imaged on the DR22A DR Translluminator to capture the fluorescent ligation product. Then the gel was post-stained in 3X Gelred solution to detect the size of the ligation product.
  • the composition of the reaction buffer that forms the reaction products of the different lanes in FIG. 9A is shown in the following table (unit: microliter).
  • the linker without redundant nucleotides (lane 9) has one extra nucleotide than the linker (lane 9)
  • the linker has a higher yield (lane 10).
  • nucleotides are connected one by one according to the principle of base pairing. ⁇ 3' direction synthesis and template complementary strand to form a 94bp product.
  • Example 9 The influence of temperature on the nick translation method to connect the 3'end of the adaptor and the 5'end of the target DNA fragment
  • the single-stranded oligonucleotide for synthesizing the linker is h5u
  • the single-stranded oligonucleotide for synthesizing the corresponding target DNA fragment is sub0fam and h3sub.
  • the sequence information is shown in Table 1 above.
  • Other materials include dNTPs, Taq DNA polymerase, E Coli DNA ligase, 1X E Coli ligase buffer, water and Gelred.
  • the operation steps were to form the linker and artificially synthesize the target DNA fragment.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer.
  • the reaction buffer included 40 pmole linker, 20 pmole target DNA, water, dNT Ps, Taq DNA polymerase, E Coli ligase and E Coli ligase buffer.
  • the mixture was incubated at 30.1°C, 33.0°C, 35.3°C, 37.9°C, 40.7°C, 43.4°C, 46°C, and 49.8°C for 30 minutes, respectively.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour. Photograph the fluorescently labeled connection product on the DR22A DR Translluminator. Then the gel was post-stained in 3X Gelred solution to detect the size of the ligation product.
  • thermostable ligase The effect of thermostable ligase on the nick translation method to ligate the 3'end of the linker and the 5'end of the target DNA fragment
  • the single-stranded oligonucleotides for synthesizing linkers are h5u and h5un, and the oligonucleotides for synthesizing corresponding target DNA fragments are sub0fam and h3sub.
  • the sequence information is shown in Table 1 above.
  • Other materials include dNTPs, Taq DNA polymerase, E Coli DNA ligase, Taq DNA ligase, 9°N DNA ligase, E Coli DNA ligase buffer, Taq DNA ligase buffer, 9°N DNA ligase buffer Liquid, water and Gelred.
  • the operation steps were to form the linker and artificially synthesize the target DNA fragment.
  • the ligation experiment was completed in 10 ⁇ L reaction buffer.
  • the first set of reaction buffers included 40 pmole linker, 20 pmole target DNA, water, dNT Ps, Taq DNA polymerase, E Coli ligase, and E Coli ligase buffer. The mixture was incubated at 40°C for 30 minutes.
  • the second set of reaction buffers includes 40 pmole linker, 20 pmole target DNA fragment, dNTPs, Taq DNA polymerase and Taq DNA ligase. The mixture was incubated at 45.1 to 66.1°C for 30 minutes.
  • the third set of reaction buffers includes 40 pmole linker, 20 pmole target DNA fragment, dNT Ps, Taq DNA polymerase and 9°N DNA ligase.
  • the mixture was incubated at 45.1 to 66.1°C for 30 minutes.
  • the final product was electrophoresed on a 15% TBE gel at 180 volts for 1 hour. Photograph the fluorescently labeled connection product on the DR22A DR Translluminator. Then the gel was post-stained in 3X Gelred solution to detect the size of the ligation product.
  • E Coli DNA ligase can connect the 5'linker to the target DNA to form a 94bp fluorescent ligation product (Figure 11A, 11B).
  • 11A is to photograph the fluorescently labeled ligation product and artificially synthesized target DNA
  • 11B is to photograph the fluorescently labeled and then stain the gel in a 3X Gelred solution, and then photograph the gelred to detect the size of the ligated product.
  • the ligation efficiency of Taq DNA ligase is not sensitive to temperature rise, and the yield at 45.1°C is slightly higher than that at 60.1°C.
  • the ligation efficiency of 9°N DNA ligase decreases significantly with increasing temperature. At 45.1°C, the ligase efficiency is ranked from high to low: E Coli DNA ligase, Taq DNA ligase, and 9° N DNA ligase ( Figure 11A).
  • thermostable DNA ligase cannot improve the ligation efficiency of the 5'end linker.
  • Example 11 The effect of pretreatment of genomic DNA on the ligation of adhesive joints
  • the single-stranded oligonucleotides of the synthetic linker are h3c1-ddN, h3c2, h3c3, h3c4, h3s0s, h3c0-ddT, h3s0s, h3s1s, h3s2s, h3s3s, h3s4s, and the sequence information is shown in Table 1 above.
  • the target is genomic DNA Coriell NA12878, and other materials include NEBNext dsDNA Fragmentase, shrimp alkaline phosphatase (rSAP), NEB Blunt TA ligase, EDTA, water, Gelred.
  • the average size of the fragmented DNA was about 220 bp ( Figure 12, lane 1), and the average size of the DNA fragments after being ligated with the adapter was 280 bp ( Figure 12, lanes 2-6).
  • DNA without dephosphorylation showed another peak at 500-600bp, which may be due to self-ligation of fragmented DNA ( Figure 12, lane 2).
  • the ligation efficiency of dephosphorylated DNA to the linker ( Figure 12, lanes 3-6) was significantly higher than that of DNA without dephosphorylation.
  • the oligonucleotides of the linker are h3c1-ddN, h3c2, h3c3, h3c4, h3s0s, h3c0ddT, h3s0s, h3s1s, h3s2s, h3s3s, and h3s4s.
  • the sequence information is shown in Table 1 above.
  • the target is free peripheral blood DNA.
  • Other materials include shrimp alkaline phosphatase, T4 ligase, NEB blunt TA ligase, KAPA ligase, NEB Q5 polymerase, dNTPs, Taq DNA polymerase, E Coli DNA ligase, uracil specific excision enzyme, water And Gelred.
  • M1 is a single blunt end joint (h3c0-ddT/h3s0s); M3, M5, M7, and M9 are a mixture of blunt end and sticky end joints: M3 (h3c1-ddN/h3s0s, h3c0-ddT/h3s0s, h3c0-ddT/h3s1s); M5(h3c2/h3s0s, h3c1-ddN/h3s0s, h3c0-ddT/h3s0s, h3c0-ddT/h3s1s, h3c0-ddT/h3s2s); M7(h3c2/h3ch3c1- ddN/h3s0s, h3c0-ddT/h3s0s, h3c0-ddT/h3s2s); M7(h3c2/h3ch3c1- ddN/h3s0s, h3c0-d
  • the first step of the ligation reaction includes dephosphorylated DNA, mixed linkers of different combinations and NEB Blunt TA ligase.
  • genomic DNA that was not dephosphorylated was reacted alone in another tube.
  • the reaction conditions were 15 minutes of incubation at 25°C.
  • the reaction mixture includes:
  • the ligated product was purified with 1.2X magnetic beads, and finally washed with 14 ⁇ l of water and collected 13 ⁇ l of purified DNA.
  • the second step ligation reaction system includes the following components.
  • the reaction conditions were incubation at 40°C for 30 minutes.
  • the reaction mixture includes:
  • the ligated product was purified with 1X magnetic beads, and finally washed with 14 ⁇ l of water and collected 13 ⁇ l of purified DNA.
  • the reaction system includes the following components:
  • the PCR amplification program is:
  • the amplified product was purified with 1X magnetic beads, and finally washed with 27 ⁇ l of water and collected 25 ⁇ l of purified DNA.
  • the main peak of free DNA library is about 310 bp ( Figure 13: lanes 1-7), and the secondary peak is 480 bp.
  • the yield of the library without dephosphorylated free DNA (lane 1) was significantly lower than the yield of the library with dephosphorylated free DNA ( Figure 13: lanes 2-6).
  • the yield of the library constructed by the mixture of blunt end and sticky end joints (M3, M5, M7, M9) was significantly higher than that of the blunt end joint M1.
  • the M5 hybrid linker has the highest library yield ( Figure 13, lane 4).
  • the present invention can use free DNA to build a library. Because the repair and complementation of target DNA are omitted, the 5'end phosphorylation and 3'end addition of A are experimental steps, which greatly improves the transformation efficiency.

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Abstract

提供了一种DNA文库构建方法，该方法包括如下步骤：(1)将DNA片段5'末端去磷酸化；(2)提供接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头；(3)使所述接头混合物中接头的5'末端与去磷酸化的DNA片段3'末端连接；(4)通过切口平移法使接头混合中接头的3'末端与所述DNA片段5'末端相连。

Description

一种DNA文库构建方法 技术领域
本发明提供涉及DNA文库的构建方法，以及使用该文库测序一个或多个靶核酸的方法及其组合物、试剂盒。
背景技术
核酸序列编码生物功能和繁殖所必需的信息。因此，对核算序列进行测序是对生物体进行研究，以及在疾病诊断和药品开发等应用中非常重要的基础技术。在医学领域，测序方法通常被用于诊断和治疗各种疾病，例如包括癌症、传染病、自身免疫疾病、遗传性疾病等。为了使测序技术能更加普及地用于各种用途，一方面需要更高通量和/或更低成本的核酸测序技术，另一方面还需要能够对更加微量的核酸进行测序。对微量核酸进行测序在某些情况下尤为重要，例如通过对母体外周血游离核酸进行测序来检测胎儿相关核酸序列，以及通过对患者外周血游离核酸测序来诊断癌症等。
目前的二代测序(next generation sequencing，NGS)基础是测序文库的构建。文库构建流程通常包括：(1)将未片段化的基因组DNA进行超声波打断，或者酶片段化处理(如使用血浆游离DNA或其它已片段化的DNA可省略此步)；(2)将DNA片段进行末端缺口补齐修复；(3)在经过末端修复的DNA片段的5’末端磷酸化，3’末端添加单碱基A(腺嘌呤)以便获得具有单个碱基粘性末端；(4)将具有粘性末端A的DNA片段与根据不同测序平台设计的接头相连(接头可带样本标签)，以获得连接产物；(5)如果是对靶向富集区域测序(如全外显子组测序或NGS panel测序)，通过接头上的各测序平台的通用引物对连接产物进行PCR，以获取足量的扩增后的DNA文库(在此步亦可引入样本标签)；(6)将扩增后的DNA文库用探针捕获法或靶向引物法进行目标区富集制备测序文库。
NGS建库方法的设计会直接影响起始DNA通过与接头连接进入文库的转化率，进而影响到最终测序文库的产量与可分析的起始DNA分子多样性(DNA complexity)。传统方法在建库中存在的普遍问题包括：DNA平末端或经过末端修复的DNA片段的3’末端添加碱基A获得的单个碱基粘性末端连接效率低，5’末端不完全的磷酸化阻止连接，3’末端非特异的添加碱基A(误加G、C、T)阻碍接头与DNA的有效AT连接，以及接头和目标DNA的自连接阻碍阻碍接头与DNA连接。5’末端3’末端由于传统建库方法中存在的连接效率较低的问题，起始DNA用量要求较高，如果起始DNA不足则需要通过PCR扩增的方法提高文库产量以便 于流程下游样本处理。但是PCR扩增会引入重复序列和错误降低测序质量。以游离DNA(cell free DNA，cfDNA)为例，1ng的cfDNA含有约330个基因组拷贝，如果使用10ng的起始DNA，最后的最大理论测序深度应该在3300，但是运用传统NGS建库方法，大部分起始DNA无法完成建库的转化率。最后在临床应用中测序有效深度一般只会是理论测序深度的一小部分。
为对微量DNA进行测序，亟需开发一种DNA文库构建方法，以提高DNA文库转化效率和质量以及测序效率。
发明内容
本发明开发了一种新型DNA测序或基因型分型文库构建方法，该方法提高了接头的多样性，从而能够与具有不同末端的天然DNA以及通过机械或酶方法片段化的DNA进行有效连接。
在一些实施方案中，本发明提供了一种DNA文库构建方法，其中所述方法包括如下步骤：(1)将DNA片段5’末端去磷酸化；(2)提供接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头；(3)使所述接头混合物中接头的5’末端与去磷酸化的DNA片段3’末端连接；(4)通过切口平移法(nick translation)使接头混合中接头的3’末端与所述DNA片段5’末端相连。
本发明构建的DNA文库可以用于测序、基因型分型等多种用途。本发明所提供的DNA文库构建方法不受任何具体测序或基因型分型平台的限制，可以广泛适用于不同的DNA分析平台。而且，本发明所提供的文库构建方法适合于针对不同长度的DNA片段构建文库。在一个实施方案中，用于本发明方法的DNA片段长度例如小于10000bp，1000bp、小于900bp、小于800bp、小于700bp、小于600bp、小于500bp、小于400bp、小于300bp、小于200bp、小于150bp、小于100bp、小于90bp、小于80bp、小于70bp、小于60bp、小于55bp、小于50bp、小于45bp、小于40bp或小于35bp。
在另一个的实施方案中，所述带有不同长度粘末端的接头的粘末端长度为1-10bp，1-9bp，1-8bp，1-7bp，1-6bp，1-5bp，1-4bp，1-3bp，1-2bp或1bp。其中，所述粘末端为5’粘末端和/或3’粘末端。在一个实施方案中，所述粘末端为随机核苷酸序列，例如为随机脱氧核苷酸序列(即在粘末端的每一位置均可为A、T、C、G或经修饰的A、T、C、G中的一种)，从而形成随机接头混合物。在另一实施方案中，所述带有不同长度粘末端的接头包括通用序列和随机序列，其中所述随机序列部分即为粘末端。在另一个实施方案中，所述接 头混合物的至少一个接头可以包含单分子标记(unique molecular barcode)和/或样本标签，其中所述单分子标记和/或样本标签可以位于接头的双链部分，也可以是仅作为单链存在，作为单链存在时，其位于与DNA片段相连端相对的另一端(即，不参与与目标DNA片段链接的一端)。对于带有粘末端的接头而言，作为单链存在的单分子标记和/或样本标签位于与粘末端相对的另一端。在另外一些实施方案中，接头混合物中至少一个接头中与DNA片段相连端相对的另一端(即，不参与与目标DNA片段链接的一端)被环形序列封闭，具体的接头形状如图1B所示。
在一个实施方案中，所述接头混合物中至少一个接头的总长度(即双链部分和单链部分之总和，单链部分可能仅存在于其中一端，也可能存在于两端)为21-53bp，21-54bp，21-55bp，21-56bp，21-57bp，22-54bp，23-55bp，24-56bp，25-57bp，21-87bp，22-88bp，23-89bp，24-90bp，25-91bp，26-92bp，27-93bp，28-94bp，29-95bp，或30-96bp。在另一个实施方案中，所述双链部分的长度为不短于5bp，不短于10bp，不短于15bp或不短于20bp。在另一个实施方案中，所述接头混合物中至少一个接头与DNA相连接的3’末端(即正义链3’末端)具有SpC3(C3间隔基团)或经修饰的核苷酸，所述经修饰的的核苷酸可以是双脱氧胸苷、双脱氧胞苷、双脱氧鸟苷或者双脱氧腺苷。
在另一个的实施方案中，所述核苷酸文库构建方法在将DNA片段5’末端去磷酸化之前，还包括使DNA进行片段化的步骤。所述片段化的方法包括超声波打断、酶片段化处理等。本发明不对片段化DNA的方法进行限定，只要能够使较大片段的DNA形成本发明方法所适合大小的DNA片段的方法均可用于本发明的方法中。在对基因组DNA等长片段DNA进行文库构建时，需要在5’末端去磷酸化之前增加片段化DNA的步骤。
在另一个实施方案中，所述DNA文库构建方法步骤(4)之后还包括步骤(5)：对与接头相连接的DNA片段进行扩增。由于所述接头混合物中的至少一个接头包含通用序列，可以针对该通用序列设计扩增引物，从而通过PCR等方法进行扩增。本发明不对DNA扩增方法进行限定，本领域已知的任何扩增方法均可适用于本发明。可使用的扩增技术的实例包括但不限于，PCR、定量PCR、定量荧光PCR(QF-PCR)、多重荧光PCR(MF-PCR)、实时PCR(RT-PCR)、原位滚环扩增(RCA)、连接酶链反应(LCR)等。在另一个实施方案中，通过在扩增引物中加入样本标签而在扩增步骤中加入该样本标签。
在另一个实施方案中，在步骤(3)中，通过使用连接酶将所述接头混合物中接头的5’末端与去磷酸化的DNA片段的3’末端相连接。本发明不意图对所使用的连接酶进行限定，只要能够将接头混合物中接头的5’末端与DNA片段3’末端相连接的任何连接酶均可 用于本发明的方法中。常用的连接酶包括但不限于：NEB Blunt TA连接酶混合物、T7连接酶、T4连接酶及它们两种或多种的混合物等。
在另一个实施方案中，在步骤(4)中，通过本领域技术人员公知的任何切口平移法(nick translation)使接头混合物中接头的3’末端与所述DNA片段5’末端相连。由于片段化DNA片段的5’末端进行了去磷酸化处理，虽然接头可以与目标DNA片段负链通过互补序列结合，但是在步骤(3)中其3’端不能与目标DNA片段的5’端形成连接，为此需要通过切口平移法来进行连接。在一个实施方案中，该步骤切口平移法混合使用Taq DNA聚合酶和E Coli连接酶。在另一个实施方案中，该步骤切口平移法的反应温度为30.0至50.0℃，35.0至45.0℃，36.0至44.0℃，37.0至43.0℃，38.0至42.0℃，39.0至41.0℃，或40℃。在另一个实施方案中，在步骤(4)反应前，还包括往反应物中添加5’端接头单链核苷酸的步骤。其中所述5’端接头单链是指，与目标DNA的5’端相连接的单链。
本发明在NGS文库构建中省略了目标DNA末端的修复补齐和5’末端磷酸化及3’末端添加A等实验步骤。运用不同长度的粘性末端混合接头明显提高了连接效率。并且通过对DNA片段进行5’末端去磷酸化和对接头3’末端的修饰降低了DNA片段及其接头本身的自连接，从而大大提升了建库转化率。
在另一些实施方案中，本发明提供了用于构建DNA文库的试剂盒，所述试剂盒包括接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头。在另一个的实施方案中，所述带有不同长度粘末端的接头的粘末端长度为1-10bp，1-9bp，1-8bp，1-7bp，1-6bp，1-5bp，1-4bp，1-3bp，1-2bp或1bp。其中，所述粘末端为5’粘末端和/或3’粘末端。在一个实施方案中，所述粘末端为随机核苷酸序列，例如为随机脱氧核苷酸序列(即在粘末端的每一位置均可为A、T、C、G，或经修改的A、T、C、G中的一种)，从而形成随机接头混合物。在另一实施方案中，所述带有不同长度粘末端的接头包括通用序列和随机序列，其中所述随机序列部分即为粘末端。在另一个实施方案中，所述接头混合物的至少一个接头可以包含单分子标记(unique molecular barcode)和/或样本标签，其中所述单分子标记和/或样本标签可以位于接头的双链部分，也可以是仅作为单链存在，作为单链存在时，其位于与DNA片段相连端相对的另一端(即，不参与与目标DNA片段链接的一端)。对于带有粘末端的接头而言，作为单链存在的单分子标记和/或样本标签位于与粘末端相对的另一端。在另外一些实施方案中，接头混合物中至少一个接头中与DNA片段相连端相对的另 一端(即，不参与与目标DNA片段链接的一端)被环形序列封闭，具体的接头形状如图1B所示。
在另一个实施方案中，所述接头混合物中至少一个接头的总长度(即双链部分和单链部分之总和，单链部分可能仅存在于其中一端，也可能存在于两端)为21-53bp，21-54bp，21-55bp，21-56bp，21-57bp，22-54bp，23-55bp，24-56bp，25-57bp，21-87bp，22-88bp，23-89bp，24-90bp，25-91bp，26-92bp，27-93bp，28-94bp，29-95bp，或30-96bp。在另一个实施方案中，所述双链部分的长度为不短于5bp，不短于10bp，不短于15bp或不短于20bp。在另一个实施方案中，所述接头混合物中至少一个接头与DNA相连接的3’末端(即正义链3’末端)具有SpC3封闭物或经修饰的核苷酸，所述经修饰的的核苷酸可以是双脱氧胸苷、双脱氧胞苷、双脱氧鸟苷或者双脱氧腺苷。
在另一个实施方案中，本发明提供的用于构建DNA文库的试剂盒还包含连接酶和/或其他试剂。所述连接酶包括但不限于：NEB Blunt TA连接酶混合物、T7连接酶、T4连接酶及它们两种或多种的混合物等。在另一个实施方案中，本发明提供的用于构建DNA文库的试剂盒还包含用于切口平移法的酶和/或其他试剂。在一个实施方案中，所述用于切口平移法的酶是Taq DNA聚合酶和E Coli连接酶。在另一个实施方案中，本发明提供的用于构建DNA文库的试剂盒还包含进行DNA扩增的酶和/或其他试剂。
发明详述
在本发明的详述中，为了解释目的阐述了很多具体的细节以使得本领域技术人员能够对公开实施方案进行理解。然而，本领域技术人员将理解的是，这些实施方案的具体细节并不构成对本发明保护范围的限定。此外，本领域技术人员可容易地理解，本发明相关描述和实施方法的具体顺序仅仅是说明性的，可对相关顺序可进行改变，但其仍在本发明所公开的实施方案的精神和范围内。
除非另外定义，本文使用的所有技术和科学术语具有与本领域所属领域普通技术人员通常理解的相同的含义。当所结合参考中的术语的定义与本发明说明书中提供的定义不同时，应以本说明书中提供的定义为准。
在整个说明书和权利要求中，下列术语采用本文明确相关的含义，除非上下文另有清楚指示。
本文所使用的短语“在一个实施方案中”不一定指相同的实施方案，尽管其可能指相同的实施方案。此外，如本文所使用的短语“在另一个实施方案中”不一定指不同的实施方 案，尽管其可能指不同的实施方案。因此，可容易地组合本发明的多个实施方案，而不脱离本发明的范围或精神。
在整个说明书中，“一个”、“一种”和“所述”的含义包括复数在内。
如本文所使用的，术语“文库”指多个核酸，例如，多个不同核酸。“DNA文库”是指多个DNA，例如多个不同的DNA。
本文中所使用的术语“DNA”可以是任何包含脱氧核糖核苷酸的聚合物，包括但不限于经过修饰的或者未经修饰的DNA。本领域的技术人员可以理解，用于本发明建库方法的DNA的来源不受特别限制，可以从任何可能的途径获得。根据本发明的实施方案，可以从样本中提取获得基因组DNA，也可以提取游离DNA，例如血浆游离DNA(cfDNA)。DNA样本可以来源于哺乳动物、植物、和微生物的至少一种。根据本发明的一些实施方案，基因组DNA可以为人类全血基因组DNA，优选为外周血单核细胞基因组DNA。在另一些实施方案中，所述DNA是人外周血中的游离DNA。
本文中所使用的术语“接头”是指能够与目标DNA片段相连接的任何DNA片段。本发明所使用的接头包括平末端接头和粘末端接头。“粘末端接头”是指预期与DNA片段相连接的一端具有突出单链的接头。所述粘末端(即，突出的单链)长度为1-10bp，1-9bp，1-8bp，1-7bp，1-6bp，1-5bp，1-4bp，1-3bp或1-2bp。所述粘末端可以是5’粘末端(即，突出的单链为5’端)或5’粘末端(即，突出的单链为3’端)。在一些实施方案中，粘末端接头中的粘末端(即，突出单链)为随机序列，即由A、T、C或G(或经修饰的A、T、C或G)随机组成的序列。例如，当粘末端为1bp时，其可以是A、T、C或G；当粘末端为2bp时，其可以是AA、AT、AC、AG、TA、TT、TC、TG、CA、CT、CC、CG、GA、GT、GC、GG；并以此类推。随机序列的粘末端可以与具有相应互补粘末端序列的目标DNA片段杂交，便于通过连接酶使接头DNA的5’端与目标DNA的3’端相连接。“平末端接头”则与“粘末端接头”相对，是指预期与DNA片段相连接的一端不具有突出单链的接头。
为描述方便的目的，本发明说明书和权利要求书中将本发明所使用的接头的两条DNA单链分别称为“5’端接头单链”(或“正义链”)和“3’端接头单链”(或“反义链”)。其中，“5’端接头单链”(或“正义链”)是指与目标DNA片段5’端相连接的单链；“3’端接头单链”(或“反义链”)是指与目标DNA片段3’端相连接的单链。在本发明实施方案中，由于对目标DNA片段的5’端进行了去磷酸化处理，5’端接头单链(正义链)不能与目标DNA片段的5’端通过连接酶进行连接。
如无特别说明，当与本发明的“接头”一起提及时，5’端是指与DNA相连接的5’端，即“3’端接头单链”(反义链)的5’端；3’端是指参与与DNA相连接的3’端，即“5’端接头单链”(正义链)的3’端。
在一些实施方案中，本发明的接头包含“通用序列”。“通用序列”是指，接头混合物中两种及以上接头中均包含的序列。与随机序列不同，通用序列一般是已知的序列。通用序列可以是单分子标记和/或样本标签。使用相同的通用序列，可以对包含通用序列的所有片段采用相似的方法或技术(例如，使用相同的引物或探针)进行相同或相似的操作和/或处理，例如，扩增、鉴定、测序、分离等。例如，在扩增步骤中，通用序列为不同目标DNA的集合提供了共同引物的结合位点。
在本发明的一个方面，本发明提供了一种DNA文库构建方法，其中所述方法包括如下步骤：(1)将DNA片段5’末端去磷酸化；(2)提供接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头；(3)使所述接头混合物中接头的5’末端与去磷酸化的DNA片段3’末端连接；(4)通过切口平移法使接头混合物中接头的3’末端与所述DNA片段5’末端相连。
通过本发明发明人的反复实验发现，通过使用包括平末端接头和带有不同长度粘末端接头的混合物，可以提高NGS文库构建尤其是微量DNA建库的转化效率。具体操作中，本发明省略了目标DNA的修复补齐，省略了5’末端磷酸化和3’末端添加A等实验步骤从而使实验变得更易于操作和质量控制。带粘性末端的混合接头大大提高了连接效率，5’末端去磷酸化解决了目标的自连接问题。与仅使用平末端接头相比，使用接头混合物能够提高DNA建库的转化效率。通过对接头3’末端进行修饰，解决接头自连接问题，还能进一步提高DNA建库的转化效率。
在本发明的另一方面，本发明还提供了用于构建DNA文库的试剂盒，所述试剂盒包括接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头。在该方面的一些实施方案中，本发明提供的用于制备DNA文库的试剂盒还包含连接酶、用于切口平移法的酶和/或其他试剂。本发明的提供的试剂盒可以用于本发明的DNA文库构建方法。
本发明提供的方法不限于特定的应用，而是广泛地应用于研究、临床、医药和其他生物学、生物化学和分子生物学。示例性用途包括遗传学和基因组学，例如鉴定突变和/或等位基因。在一些实施方案中，本发明的方法可以用于检测胎儿的基因是否存在异常等。本发明提供的方法还可用于传染病领域，例如鉴定病毒、细菌、真菌等传染源，测定病毒类型和家族等，以及鉴定突变和/或等位基因。在一些实施方案中，所述方法可用于肿瘤学和肿瘤学 相关领域。在肿瘤学领域的具体和非限制的说明性实例为鉴定与癌症相关的遗传和/或基因组突变、等位基因等。在一些实施方案中，所述技术可用于癌症筛查、癌症诊断、癌症预后以及选择癌症治疗方法等。
附图说明
本发明的上述和/或其他的特征、方面和优点从结合下面附图对实施方式的描述中将变得明显和容易理解，其中：
图1A显示了本发明文库构建方法示意图。从左至右分别包含的步骤为：提供DNA片段；DNA片段5’端去磷酸化；去磷酸化的DNA片段3’端与接头5’端相连接；DNA片段的5’端与接头3’端通过切口平移法连接上；通过PCR扩增并引入样本标签。图1B是本发明部分具有粘末端的接头的形状示意图。
图2显示了不同浓度的连接酶对连接效率的影响。
图3显示了不同长度的接头粘性末端对连接效率的影响。
图4显示了接头DNA 3’末端修饰对连接效率的影响。
图5显示了双脱氧胸苷末端修饰对接头自连接的影响。
图6显示了带双脱氧胸苷3’末端修饰接头的连接效率。
图7显示了不同连接酶对3’端粘性混合接头的连接效率影响。
图8显示了不同连接酶对5’端粘性混合接头的连接效率影响。
图9显示了通过切口平移法可以连接接头3’端和目标DNA5’端。
图10显示了温度对切口平移法连接接头3’末端与目标DNA片段5’末端的影响。其中图中所显示的数字为温度(℃)。
图11显示了热稳定连接酶对切口平移法连接接头3’末端与目标DNA片段5’末端的影响。其中图中所显示的数字为温度(℃)。
图12显示了基因组DNA的预处理对粘性接头连接的影响。
图13显示了使用本发明方法对微量游离DNA进行建库的结果。
实施例
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商的，均为可以通过市购获得的常规产品。
在以下实施例中，本发明使用了以下DNA核苷酸序列：
表1.本发明实施例所使用的核苷酸序列
Figure PCTCN2020115275-appb-000001
Figure PCTCN2020115275-appb-000002
Figure PCTCN2020115275-appb-000003
在上述表格序列以及本发明说明书中，“ideoxyU”表示脱氧苷，“N”表示简并碱基(例如，该位置可以是A、T、C或G)，“*”表示硫代磷酸酯键，“phos”表示端磷酸化修饰，“SpC3”表示C3间隔基团，“Biosg”是指生物素标记，“6-FAM”是指6-羧基荧光素，“ddT”表示双脱氧胸苷，“ddN”表示双脱氧胸苷、双脱氧胞苷、双脱氧鸟苷或者双脱氧腺苷。
实施例1：不同浓度的连接酶对连接效率的影响
实验材料：寡核苷酸链h 3c0、h3control、subm1和subnofam，序列信息见上表1。其他实验材料包括NEB T4连接酶、NEB Blunt TA连接酶混合物、无DNAse/RNAse水、Gelred。
实验仪器：热循环仪、凝胶电泳、Agilent Bioanalyzer 2100
实验步骤：人工合成寡核苷酸链h3c0、h3control、subm1和subnofam。对所述寡核苷酸链进行复性，分别生成接头和目标DNA。复性使用以下操作步骤：将每个寡核苷酸沉淀物短暂离心，溶解于双重缓冲液(100mM醋酸钾；30mM HEPES，pH7.5)。以等摩尔量将2个寡核苷酸聚合在一起。将混合的寡核苷酸加热至94℃ 2分钟并逐渐在室温下冷却。
连接实验在10μL反应缓冲液中完成，反应缓冲液包括T4 DNA连接酶缓冲液、10pmole的DNA片段、50pmole接头、400，800，或者1200单位T4 DNA连接酶或1800单位NEB Blunt TA连接酶和水。将反应混合物在25℃下温育15分钟，将最终产物稀释5倍并在50伏的5％TBE凝胶上电泳105分钟，并使用Agilent Bioanalyzer 2100进行定量。
实验结果：没有连接酶的情况下，接头和目标DNA不能形成连接产物(图2，第2道)。有连接酶的情况下接头和DNA片段能够形成连接产物，连接的产率取决于T4DNA连接酶的使用量(400至1200U图2，泳道3-5)，NEB Blunt TA连接酶具有最高产量(图2，泳道6)。
实验结论：h3c0和h3control的双链体可以作为TA连接对照实验。在3’末端具有SpC3封闭物的接头的连接效率高度依赖于连接酶的使用量，NEB Blunt TA连接酶具有最高连接效率。
实施例2：不同长度的接头粘性末端对连接效率的影响
实验材料：合成接头的单链寡核苷酸链为h 3c0、h 3s0、h3s1、h3s2、h3s3，合成目标DNA片段的单链寡核苷酸链为subnofam、sub0、sub5p1、sub5 p2、sub5p3和submn1，序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物、无DNAse/RNAse水、Gelred。
实验仪器：热循环仪、凝胶电泳、Agilent Bioanalyzer 2100
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，缓冲液包括3.5μL NEB Blunt TA连接酶混合物、5pmole的 DNA片段、25pmole的接头。将混合物在25℃下温育15分钟。将最终产物稀释5倍并通过Agilent Bioanalyzer 2100分析。
实验结果：在该实验中使用的所有4个接头在3’末端具有SpC3封闭物。基于Bioanalyzer 2100的定量，当5’突出端的长度为0、1、2和3个碱基时，连接产物的百分比为4.5％、11.0％、95.2％、83.8％(图3)。
实验结论：突出长度大于1bp的粘性接头连接效率(84％)显著高于平端接头(5％)。当接头的3’末端带有SpC3封闭物而5’末端的突出长度为1bp或者0bp时，接头的连接效率小于11％(图3)。
实施例3：接头DNA 3’末端修饰对连接效率的影响
实验材料：合成接头的寡核苷酸单链为h3c0ddT、h 3c0、h 3s0、h3s1、h3s2、h3s3、h3control，合成相应人工目标DNA的寡核苷酸单链为subnofam、sub 0、sub5p1、sub5p2、sub5p3，序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物、无DNAse/RNAse水、Gelred。
实验仪器：热循环仪、凝胶电泳、Agilent Bioanalyzer 2100
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括3.5μL NEB Blunt TA连接酶混合物、5pmole的目标DNA片段、25pmole的接头和水。将混合物在25℃下温育15分钟。将最终产物稀释5倍并通过Agilent Bioanalyzer 2100分析。
实验结果：在该实验中使用的前五个接头的3’末端具有双脱氧胸苷(ddT)，当5’突出端的长度为0、1、2、3和4bp时，连接分子的百分比为84.0％、73.2％、75.2％、86.5％和66.8％(图4：1-5)。在该实验中使用的后五个接头的3’末端具有SpC3封闭物(图4：6-10)。当5’突出端的长度为0、1、2、3和4bp时，连接分子的百分比为4.2％、1.6％、85.3％、68.4％和0.0％(图4)。
实验结论：接头的3’末端修饰对连接效率有显着影响。3’末端双脱氧胸苷具有比3’末端SpC3封闭物显着更高的连接效率。本实验中平末端接头的链接效率与3bp粘末端相近有几个原因，3’末端具有双脱氧胸苷的修饰提高了连接效率，使用高浓度的连接酶如NEB Blunt TA连接酶混合物。由于打断后的DNA末端可以是平端或者粘端，本发明的接头混合物可以高效与各种断头的目标DNA相连，并且可以降低连接酶的浓度以减低实验成本。
实施例4：双脱氧胸苷末端修饰对接头自连接的影响
实验材料：合成接头的寡核苷酸单链为bioh3cm1、h3c0ddT、h 3s0s、h3s1s、h3s2s、h3s3s，序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物、无DNAse/RNAse水、Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统。
实验步骤：如实验1中所描述操作步骤形成接头。连接实验在10μL反应缓冲液中完成，反应缓冲液包括3.5μL NEB Blunt TA连接酶混合物、25pmole的接头和水。在该实验中测试了两组接头。第一组接头的3’末端没有双脱氧胸苷(ddT)，第二组接头的3’末端带有双脱氧胸苷。接头的5’末端有0、1、2、3、4碱基的突出端。所有反应缓冲液在25℃下温育15分钟。最终产物在15％TBE凝胶中用电泳分析。
实验结果：在该实验中使用的前五个接头在3’末端不具有双脱氧胸苷。在20bp和32、33、34、35和36bp的条带处可以观察到变性的寡核苷酸，在52、53、54和55bp条带处观察到它们自连接的寡核苷酸(图5：泳道1-5)。在该实验中使用的第二组五个接头在3’末端具有双脱氧胸苷，在21bp和32、33、34、35和36bp的条带处可以观察到变性的寡核苷酸，但是没有明显的自连接产物(图5：泳道6-10)
实验结论：接头的3’末端双脱氧胸苷修饰在可检测范围内阻断了接头的自连接。
实施例5：带双脱氧胸苷3’末端修饰接头的连接效率
实验材料：合成接头的寡核苷酸单链为bioh3cm1、h3c0ddT、h 3s0s、h3s1s、h3s2s、h3s3s、h3s4s，合成相应目标DNA片段的寡核苷酸单链为subfam、sub 0spc3、sub5p1、sub5p2、sub5p3、sub5p4。序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物，无DNAse/RNAse水，Gelred。
实验仪器：热循环仪，XCell SureLock电泳系统，凝胶成像系统，DR22A DR Translluminator。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括5μL NEB Blunt TA连接酶混合物、25pmole接头、5pmole DNA片段和水。在该实验中比较了两组接头。第一组接头不带有双脱氧胸苷3’末端修饰，第二组接头带有双脱氧胸苷3’末端修饰。两组接头的5’末端有0、 1、2、3、4个碱基的突出端。目标DNA片段的负链(3’端与接头相连的链)5’末端带有荧光蛋白FAM，而3’末端有0、1、2、3、4个碱基的突出端。将混合物在25℃下温育15分钟。最终产品在15％TBE凝胶上以180伏电压电泳电泳1小时。将凝胶在DR22A DR Translluminator上拍摄荧光标记的连接产物。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：在没有连接酶的情况下，带有荧光的的目标DNA片段(36bp)不能够与接头形成连接产物(图6A：泳道11)。对于不带有双脱氧胸苷3’末端修饰的接头，具有2、3、4、5bp的5’突出端的接头可以与目标DNA片段分别形成69、70、71、72bp的连接产物(图6A：泳道2-5)。而只有1bp 5’突出端并且不带有双脱氧胸苷3’末端修饰的接头未能与目标DNA形成连接产物(图6A：泳道1)。带有双脱氧胸苷3’末端修饰的所有接头都可以与目标DNA片段形成连接产物(图6A：泳道6-10)。它们的连接效率也高于不带有双脱氧胸苷3’末端修饰的接头。大部分不带有双脱氧胸苷3’末端修饰的接头都形成了自连接产物(图6B：泳道1-5)，而未能与目标DNA形成带有荧光的连接产物，这与实验4的结果一致。
实验结论：3’末端双脱氧胸苷修饰显著地提高了接头与DNA片段的连接效率。
实施例6：不同连接酶对3’端粘性混合接头的连接效率影响
实验材料：合成接头的寡核苷酸单链为h3c0ddT、h3s0S、h3s1s、h3s2s、h3s3s、h3s4s，合成相应目标DNA片段的寡核苷酸单链为subfam、sub0spc3、sub5p1、sub5p2、sub5p3、sub5p4，寡核苷酸序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物，T7连接酶、T4连接酶、无DNAse/RNAse水和Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、DR22A DR Translluminator。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括不同连接酶(连接酶及其用量见附图7A和B，每一种连接酶后的数字为连接酶的量，单位为微升)和连接酶缓冲液混合物、50pmole接头混合物、5pmole的目标DNA混合物和水。接头混合物由具有不同3’突出端的接头以等摩尔比混合形成，目标DNA混合物由具有不同5’突出端的目标DNA以等摩尔比混合形成。在该实验中测试的连接酶包括NEB Blunt TA连接酶、T7连接酶、T4连接酶、T7连接酶和T4连接酶的组合。最终产物在15％TBE凝胶上以180伏电压电泳1小时。然后将凝胶 在DR22A DR Translluminator拍摄荧光标记的连接产物。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：在没有连接酶的情况下，带有荧光的的目标DNA(36bp)不能够与接头形成连接产物(图7A：泳道9)。T4连接酶的连接效率(图7A：泳道4)与NEB Blunt TA连接酶的效率相当(图7A：泳道1和2)。单独使用T7连接酶(图7A：泳道3)的连接效率最低，T7和T4连接酶的组合降低了T4的连接效率(图7A：泳道7)。从Gelred染色的图片中可以发现使用T7连接酶产生了一些较大的非特异性连接产物(图7B，泳道3、5、6、7、8)。
实验结论：带有双脱氧胸苷3’末端修饰接头在使用NEB Blunt TA连接酶或者T4连接酶的连接效率无明显改变，而粘性末端特异性T7连接酶并没有提高带有双脱氧胸苷3’末端修饰接头的连接效率。
实施例7：不同连接酶对5’端粘性混合接头的连接效率影响
实验材料：合成接头的寡核苷酸单链为h3c1、h3c2、h3c3、h3c4、h 3s0s，合成相应DNA片段的寡核苷酸单链为sub0spc3、sub3p1、sub3p2、sub3p3、sub3p4。序列信息见上表1。其他实验材料包括NEB Blunt TA连接酶混合物、T7连接酶、T4连接酶、无DNAse/RNAse水、Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括5μL不同连接酶和连接酶缓冲液混合物、50pmole接头混合物、5pmole目标DNA混合物和水。接头混合物由具有不同5’突出端的接头以等摩尔比混合形成，目标DNA混合物由具有不同3’突出端的目标DNA以等摩尔比混合形成。在该实验中测试的连接酶包括NEB Blunt TA连接酶、T7连接酶、T4连接酶、T7连接酶和T4连接酶的组合。最终产物在15％TBE凝胶上以180伏电压电泳1小时，然后将凝胶在3X Gelred溶液中后染色以检测是否有连接产物和连接产物的大小。
实验结果：在没有连接酶的情况下，目标DNA(36bp)不能够与接头形成连接产物(图8：泳道10)。具有1、2、3、4bp的5’粘性末端的接头可以与目标DNA形成连接产物(图8，泳道3-6)。T7/T4连接酶的组合产生了一些较大的非特异性连接产物(图8，泳道7-9)。
实验结论：NEB Blunt TA连接酶对于5’端粘性混合接头的连接效率优于T7/T4组合连接酶的连接效率。
实施例8：切口平移法连接接头3’端和目标DNA5’端
实验材料：合成接头的寡核苷酸单链为h5u和h5un，合成相应DNA片段的寡核苷酸单链为sub0fam和h3sub。序列信息见上表1。其他实验材料包括dNTPs，Taq DNA聚合酶，E Coli DNA连接酶，1X E Coli连接酶缓冲液，水，Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、DR22A DR Translluminator。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括40pmole接头、20pmole目标DNA、水、dNT Ps、Taq DNA聚合酶、E Coli连接酶和E Coli连接酶缓冲液。混合物在40℃下温育30分钟。最终产物在15％TBE凝胶上以180伏电压电泳1小时。将凝胶在DR22A DR Translluminator上成像以捕获具有荧光的连接产物。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。形成图9A中不同泳道反应产物的反应缓冲液组成如下表所示(单位为微升)。
Figure PCTCN2020115275-appb-000004
Figure PCTCN2020115275-appb-000005
实验结果：在没有聚合酶和连接酶的情况下，虽然接头可以与目标DNA片段负链通过互补序列结合，但是5’端接头单链(58bp)不能与目标DNA片段正链(36bp)的5’端相连形成连接产物(图9A：泳道5)。单独使用Taq DNA聚合酶或者E Coli连接酶也无法形成连接产物(图9A：泳道6和7)。混合使用Taq DNA聚合酶、E Coli连接酶和dNTPs可以形成94bp带荧光的连接产物(图9A：泳道9和10)，没有冗余核苷酸的接头(泳道9)比具有一个额外核苷酸的接头具有更高的产量(泳道10)。只有Taq DNA聚合酶和dNTPs的情况下未能形成带荧光的连接产物，但是可以观察到一个94bp的条带(图9B：泳道8)，这是因为当接头连接到目标DNA互补链上面后，由于Taq DNA聚合酶的5’→3’聚合酶活性，具有荧光的目标DNA被水解，同时在Taq DNA聚合酶的作用下，按照碱基配对的原则，逐个连上核苷酸，由5’→3’方向合成与模板互补链形成一个94bp的产物。
实验结论：混合使用Taq DNA聚合酶，E Coli DNA连接酶，和dNTPs能够高效的连接接头的3’末端与目标DNA片段5’末端。
实施例9：温度对切口平移法连接接头3’末端与目标DNA片段5’末端的影响
实验材料：合成接头的寡核苷酸单链为h5u，合成相应目标DNA片段的寡核苷酸单链为sub0fam和h3sub。序列信息见上表1。其他材料包括dNTPs、Taq DNA聚合酶、E Coli DNA连接酶、1X E Coli连接酶缓冲液、水和Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、DR22A DR Translluminator。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，反应缓冲液包括40pmole接头、20pmole目标DNA、水、dNT Ps、Taq DNA聚合酶、E Coli连接酶和E Coli连接酶缓冲液。将混合物分别在30.1℃、33.0℃、35.3℃、37.9℃、40.7℃、43.4℃、46℃和49.8℃温育30分钟。最终产物在15％TBE凝胶上以180伏电压电泳1小时。将凝胶在DR22A DR Translluminator拍摄荧光标记的连接产物。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：混合使用Taq DNA聚合酶、E ColiDNA连接酶和dNTPs在温度30.1至49.8℃都可以把5’接头与目标DNA连接，当温度高于37.9℃时，连接产率没有明显改善，而一些非特异性的连接产物有所增加(图10)。
实验结论：切口平移法连接5’末端接头的最佳温度为37至44℃。
实验10：热稳定连接酶对切口平移法连接接头3’末端与目标DNA片段5’末端的影响
实验材料：合成接头的寡核苷酸单链为h5u和h5un，合成相应目标DNA片段的寡核苷酸为sub0fam和h3sub。序列信息见上表1。其他材料包括dNTPs、Taq DNA聚合酶、E Coli DNA连接酶、Taq DNA连接酶、9°N DNA连接酶、E Coli DNA连接酶缓冲液、Taq DNA连接酶缓冲液、9°N DNA连接酶缓冲液、水和Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、DR22A DR Translluminator。
实验步骤：如实验1中所描述操作步骤形成接头和人工合成目标DNA片段。连接实验在10μL反应缓冲液中完成，第一组反应缓冲液包括40pmole接头、20pmole目标DNA、水、dNT Ps、Taq DNA聚合酶、E Coli连接酶、E Coli连接酶缓冲液。混合物在40℃下温育30分钟。第二组反应缓冲液包括40pmole接头、20pmole目标DNA片段、dNTPs、Taq DNA聚合酶和Taq DNA连接酶。将混合物在45.1至66.1℃温育30分钟。第三组反应缓冲液包括40pmole接头、20pmole目标DNA片段、dNT Ps、Taq DNA聚合酶和9°N DNA连接酶。将混合物在45.1至66.1℃温育30分钟。最终产物在15％TBE凝胶上以180伏电压电泳1小时。将凝胶在DR22A DR Translluminator拍摄荧光标记的连接产物。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：E Coli DNA连接酶可以把5’接头与目标DNA连接形成94bp带荧光的连接产物(图11A，11B)。其中，图11A为拍摄荧光标记的连接产物和人工合成目标DNA，11B是拍摄荧光标记然后将凝胶在3X Gelred溶液中后染色，然后以拍摄Gelred检测连接产物的大小。Taq DNA连接酶的连接效率对温度的升高不敏感，在45.1℃时的产率稍高于60.1℃时的产率。9°N DNA连接酶的连接效率随温度升高明显降低。在45.1℃时候连接酶的效率由高到低的排序是：E Coli DNA连接酶、Taq DNA连接酶、9°N DNA连接酶(图11A)。
实验结论：热稳定DNA连接酶不能提高5’端接头的连接效率。
实施例11：基因组DNA的预处理对粘性接头连接的影响
实验材料：合成接头的寡核苷酸单链为h3c1-ddN、h3c2、h3c3、h3c4、h 3s0s、h3c0-ddT、h 3s0 s、h3s1s、h3s2s、h3s3s、h3s4s，序列信息见上表1。目标为基因组DNA Coriell NA12878，其他材料包括NEBNext dsDNA Fragmentase、虾碱性磷酸酶(rSAP)、NEB Blunt TA连接酶、EDTA、水、Gelred。
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、Zymogen凝胶纯化系统。
实验步骤：
(1)通过以下方案将coriell DNA片段化：
DNA NA12878 2μg 6μl
10X片段酶反应缓冲液：2μl
无菌水：10μl
在37℃孵育35分钟
加入5μl 0.5M EDTA以终止反应。
(2)琼脂糖凝胶尺寸选择/分析：将样品直接加载到3％琼脂糖凝胶上以100伏电压电泳1小时，并选择120-150、150-180、180-210、210-240、240-270、270-300b片断的DNA进行凝胶纯化。
(3)如实验1中所描述操作步骤形成接头，并以等摩尔比混合以产生接头混合物。片断化的基因组DNA在37℃下温育15分钟，随后65℃5分钟，进行DNA去磷酸化。连接反应包括去磷酸化的DNA、不同组合的混合接头和NEB Blunt TA连接酶。作为对照，没有去磷酸化的基因组DNA在另外的管中单独反应。最终产物在15％TBE凝胶上以180伏电压电泳1小时。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：片段化的DNA的平均大小约为220bp(图12，泳道1)，与接头连接后的DNA片段平均大小为280bp(图12，泳道2-6)。没有去磷酸化的DNA在500-600bp出现了另外一个峰值，可能是由于片段化DNA自连接形成(图12，泳道2)。去磷酸化的DNA与接头的连接效率(图12，泳道3-6)明显高于没有去磷酸化的DNA连接效率。
实验结论：基因组DNA的去磷酸化预处理显着降低了目标DNA之间的自身连接问题，提高了与接头的连接效率。
实验12：微量游离DNA建库
实验材料：接头的寡核苷酸为h3c1-ddN、h3c2、h3c3、h3c4、h3s0s、h3c0ddT、h3s0s、h3s1s、h3s2s、h3s3s和h3s4s，序列信息见上表1。目标为外周血游离DNA。其他材料包括虾碱性磷酸酶、T4连接酶、NEB blunt TA连接酶、KAPA连接酶、NEB Q5聚合酶、dNTPs、Taq DNA聚合酶、E Coli DNA连接酶，、尿嘧啶特异性切除酶、水和Gelred.
实验仪器：热循环仪、XCell SureLock电泳系统、凝胶成像系统、Ampure XP磁珠和Qubit DNA定量仪。
实验步骤：
(1)如实验1中所描述操作步骤形成接头，并以等摩尔比混合以产生接头混合物。复性后的接头中，M1为单一的平端接头(h3c0-ddT/h3s0s)；M3、M5、M7、M9是平端与粘端接头混合物：M3(h3c1-ddN/h3s0s，h3c0-ddT/h3s0s，h3c0-ddT/h3s1s)；M5(h3c2/h3s0s，h3c1-ddN/h3s0s，h3c0-ddT/h3s0s，h3c0-ddT/h3s1s，h3c0-ddT/h3s2s)；M7(h3c2/h3s0s，h3c2/h3s0s，h3c1-ddN/h3s0s，h3c0-ddT/h3s0s，h3c0-ddT/h3s1s，h3c0-ddT/h3s2s，h3c0-ddT/hs3s)；M9(h3c4/h3s0s，h3c2/h3s0s，h3c2/h3s0s，h3c1-ddN/h3s0s，h3c0-ddT/h3s0s，h3c0-ddT/h3s1s，h3c0-ddT/h3s2s，h3c0-ddT/hs3s，h3c0-ddT/h3s4s)。
(2)在如下反应混合物中将游离DNA在37℃下温育15分钟并随后在65℃温育5分钟去磷酸化：
Figure PCTCN2020115275-appb-000006
8μl游离DNA(约1ng)
Figure PCTCN2020115275-appb-000007
1μl虾碱性磷酸酶
Figure PCTCN2020115275-appb-000008
1μl 10X虾碱性磷酸酶缓冲液
(3)第一步连接反应包括去磷酸化的DNA、不同组合的混合接头和NEB Blunt TA连接酶。作为对照，没有去磷酸化的基因组DNA在另外的管中单独反应。反应条件为25℃下温育15分钟。反应混合物包括：
Figure PCTCN2020115275-appb-000009
10μl去磷酸化的DNA
Figure PCTCN2020115275-appb-000010
2.5μl接头或水(M1、M3、M5、M7或M9)
Figure PCTCN2020115275-appb-000011
12.5μl 2X NEB blunt TA连接酶混合物
连接后的产物用1.2X磁珠纯化，最后用14μl的水冲洗并收集13μl纯化DNA。
(4)第二步连接反应体系包括包括以下成分。反应条件为40℃下温育30分钟。反应混合物包括：
Figure PCTCN2020115275-appb-000012
13μl纯化DNA
Figure PCTCN2020115275-appb-000013
h5u接头10uM：2μl
Figure PCTCN2020115275-appb-000014
dNTPs 2mM：1μl
Figure PCTCN2020115275-appb-000015
Taq DNA聚合酶：1μl
Figure PCTCN2020115275-appb-000016
E Coli DNA连接酶：1μl
Figure PCTCN2020115275-appb-000017
尿嘧啶特异性切除酶：1μl
Figure PCTCN2020115275-appb-000018
10X E Coli连接酶缓冲液：2μl
连接后的产物用1X磁珠纯化，最后用14μl的水冲洗并收集13μl纯化DNA。
(5)PCR扩增。反应体系包括以下成分：
Figure PCTCN2020115275-appb-000019
13μl纯化DNA
Figure PCTCN2020115275-appb-000020
扩增引物PHS-P7-001-01/HS_P5 2.5μM 2μl
Figure PCTCN2020115275-appb-000021
Q5聚合酶混合物：15μl
PCR扩增程序为：
1)98℃，30秒
2-3重复10次
2)98℃，10秒
3)65℃，75秒
4)65℃，5分钟
5)4℃，暂停
扩增后的产物用1X磁珠纯化，最后用27μl的水冲洗并收集25μl纯化DNA。
(6)最终产物用Qubit定量，并在15％TBE凝胶上以180伏电压电泳1小时。然后将凝胶在3X Gelred溶液中后染色以检测连接产物的大小。
实验结果：游离DNA建库后的主峰在310bp左右(图13：泳道1-7)，次峰在480bp。没有去磷酸化的游离DNA的文库(泳道1)产量明显低于去磷酸化游离DNA的文库产量(图13：泳道2-6)。平端与粘端接头混合物(M3、M5、M7、M9)构建的文库产量明显高于平端接头M1。M5混合接头具有最高的文库产量 (图13，泳道4)。
实验结论：本发明可以使用游离DNA建库，由于省略了目标DNA的修复补齐，5’末端磷酸化和3’末端添加A等实验步骤，大大提高了转化效率。
尽管所述技术已经结合具体的示例性实施方案进行描述，应理解的是如所要求的本发明不应被过度限制在这样的具体实施方案中。实际上，对本领域技术人员显而易见的所述用于实施本发明的模式的多种修饰意在包含在随附权利要求的范围内。
此外，除上下文另有所指外，以单数形式出现的词包括复数形式，反之亦然。
Figure PCTCN2020115275-appb-000022
Figure PCTCN2020115275-appb-000023
Figure PCTCN2020115275-appb-000024
Figure PCTCN2020115275-appb-000025
Figure PCTCN2020115275-appb-000026
Figure PCTCN2020115275-appb-000027
Figure PCTCN2020115275-appb-000028
Figure PCTCN2020115275-appb-000029
Figure PCTCN2020115275-appb-000030
Figure PCTCN2020115275-appb-000031
Figure PCTCN2020115275-appb-000032
Figure PCTCN2020115275-appb-000033
Figure PCTCN2020115275-appb-000034
Figure PCTCN2020115275-appb-000035

Claims (19)

  1. 一种DNA文库构建方法，其特征在于包括如下步骤：(1)将DNA片段5’末端去磷酸化；(2)提供接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头；(3)使所述接头混合物中接头的5’末端与去磷酸化的DNA片段3’末端连接；(4)通过切口平移法使接头混合物中接头的3’末端与所述DNA片段5’末端相连。
  2. 权利要求1所述的DNA文库构建方法，其中所述方法在步骤(1)将DNA片段5’末端去磷酸化之前，还包括使DNA进行片段化的步骤。
  3. 权利要求1或2所述的DNA文库构建方法，其中所述方法在步骤(4)通过切口平移法使接头混合物中接头的3’末端与所述DNA片段5’末端相连之后，还包括步骤(5)：对与接头相连接的DNA片段进行扩增。
  4. 权利要求1所述的DNA文库构建方法，其中所述带有不同长度粘末端的接头的粘末端为5’粘末端和/或3’粘末端，且其长度为1-10bp，1-9bp，1-8bp，1-7bp，1-6bp，1-5bp，1-4bp，1-3bp，1-2bp或1bp。
  5. 权利要求1所述的DNA文库构建方法，其中所述带有不同长度粘末端的接头的粘末端为随机核苷酸序列。
  6. 权利要求1所述的DNA文库构建方法，其中所述接头混合物中至少一个接头可以包含单分子标记和/或样本标签。
  7. 权利要求1所述的DNA文库构建方法，其中所述接头混合物中至少一个接头中与DNA片段相连端相对的另一端被环形序列封闭。
  8. 权利要求1所述的DNA文库构建方法，其中所述接头混合物中至少一个接头的总长度为21-53bp，21-54bp，21-55bp，21-56bp，21-57bp，22-54bp，23-55bp，24-56bp，25-57bp，21-87bp，22-88bp，23-89bp，24-90bp，25-91bp，26-92bp，27-93bp，28-94bp，29-95bp，或30-96bp。
  9. 权利要求1所述的DNA文库构建方法，其中所述接头混合物中至少一个接头与DNA相连接的3’末端具有SpC3封闭物或经修饰的核苷酸，所述经修饰的核苷酸可以是双脱氧胸苷、双脱氧胞苷、双脱氧鸟苷或者双脱氧腺苷。
  10. 权利要求1所述的DNA文库构建方法，其中在步骤(3)中，通过使用连接酶将所述接头混合物中接头的5’末端与去磷酸化的DNA片段的3’末端相连接。
  11. 权利要求1所述的DNA文库构建方法，其中在步骤(4)反应前，还包括往反应物中添加5’端接头单链核苷酸的步骤，其中所述5’端接头单链是指与目标DNA的5’端相连接的单链。
  12. 用于构建DNA文库的试剂盒，所述试剂盒包括接头混合物，所述接头混合物包括平末端接头和带有不同长度粘末端的接头。
  13. 权利要求12的试剂盒，其中所述带有不同长度粘末端的接头的粘末端为5’粘末端和/或3’粘末端，其长度为1-10bp，1-9bp，1-8bp，1-7bp，1-6bp，1-5bp，1-4bp，1-3bp，1-2bp或1bp。
  14. 权利要求12所述的试剂盒，其中所述带有不同长度粘末端的接头的粘末端为随机核苷酸序列。
  15. 权利要求12所述的试剂盒，其中所述接头混合物的至少一个接头可以包含单分子标记和/或样本标签。
  16. 权利要求12所述的试剂盒，其中所述接头混合物中至少一个接头中与DNA片段相连端相对的另一端被环形序列封闭。
  17. 权利要求12所述的试剂盒，其中所述接头混合物中至少一个接头的总长度为21-53bp，21-54bp，21-55bp，21-56bp，21-57bp，22-54bp，23-55bp，24-56bp，25-57bp，21-87bp，22-88bp，23-89bp，24-90bp，25-91bp，26-92bp，27-93bp，28-94bp，29-95bp，或30-96bp。
  18. 权利要求12所述的试剂盒，其中所述接头混合物中至少一个接头与DNA相连接的3’末端具有SpC3封闭物或经修饰的核苷酸，所述经修饰的的核苷酸可以是双脱氧胸苷、双脱氧胞苷、双脱氧鸟苷或者双脱氧腺苷。
  19. 权利要求12所述的试剂盒，其中所述试剂盒还包含连接酶、用于切口平移法的酶和/或用于DNA扩增的酶。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113817804A (zh) * 2021-09-22 2021-12-21 上海金匙医学检验实验室有限公司 一种测序文库自连接头消除的方法及应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110565174B (zh) * 2019-09-17 2022-09-30 北京博昊云天科技有限公司 一种dna文库构建方法
CN111254198A (zh) * 2020-03-09 2020-06-09 苏州宏元生物科技有限公司 一种尿路上皮癌诊断和预后评估试剂盒
CN111394436B (zh) * 2020-03-26 2023-12-08 天昊基因科技(苏州)有限公司 一种dna接头及其制备方法和应用
CN114807125B (zh) * 2022-05-20 2023-09-12 纳昂达(南京)生物科技有限公司 测序文库接头、测序文库及其构建方法、提升ngs建库连接效率的方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9023769B2 (en) * 2009-11-30 2015-05-05 Complete Genomics, Inc. cDNA library for nucleic acid sequencing
CN105525357A (zh) * 2014-09-30 2016-04-27 深圳华大基因股份有限公司 一种测序文库的构建方法及试剂盒和应用
CN106459968A (zh) * 2014-01-31 2017-02-22 斯威夫特生物科学股份有限公司 用于加工dna底物的改进方法
CN108166067A (zh) * 2018-01-02 2018-06-15 上海美吉生物医药科技有限公司 一种新型dna建库方法及其应用
CN108166068A (zh) * 2018-01-02 2018-06-15 上海美吉生物医药科技有限公司 一种新型dna建库试剂盒及其应用
CN110565174A (zh) * 2019-09-17 2019-12-13 北京博昊云天科技有限公司 一种dna文库构建方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19849348A1 (de) * 1998-10-26 2000-04-27 Univ Ludwigs Albert Linear Amplification mediated PCR (=LAM PCR)
EP2235217B1 (en) * 2008-01-09 2016-04-20 Life Technologies Corporation Method of making a paired tag library for nucleic acid sequencing
WO2012166647A1 (en) * 2011-05-27 2012-12-06 Life Technologies Corporation Methods for manipulating biomolecules
CN105950612B (zh) * 2016-07-08 2019-06-21 北京全式金生物技术有限公司 一种高效的dna接头连接方法
EP3692161A4 (en) * 2016-11-11 2022-05-25 Psomagen, Inc. METHODS AND SYSTEM FOR FRAGMENT ARRANGEMENT AND SEQUENCE IDENTIFICATION
CN108048915A (zh) * 2017-12-01 2018-05-18 北京科迅生物技术有限公司 用于ctDNA文库构建的接头混合物、包括其的试剂盒及应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9023769B2 (en) * 2009-11-30 2015-05-05 Complete Genomics, Inc. cDNA library for nucleic acid sequencing
CN106459968A (zh) * 2014-01-31 2017-02-22 斯威夫特生物科学股份有限公司 用于加工dna底物的改进方法
CN105525357A (zh) * 2014-09-30 2016-04-27 深圳华大基因股份有限公司 一种测序文库的构建方法及试剂盒和应用
CN108166067A (zh) * 2018-01-02 2018-06-15 上海美吉生物医药科技有限公司 一种新型dna建库方法及其应用
CN108166068A (zh) * 2018-01-02 2018-06-15 上海美吉生物医药科技有限公司 一种新型dna建库试剂盒及其应用
CN110565174A (zh) * 2019-09-17 2019-12-13 北京博昊云天科技有限公司 一种dna文库构建方法

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CN113817804A (zh) * 2021-09-22 2021-12-21 上海金匙医学检验实验室有限公司 一种测序文库自连接头消除的方法及应用
CN113817804B (zh) * 2021-09-22 2024-03-08 上海金匙医学检验实验室有限公司 一种测序文库自连接头消除的方法及应用

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