CN109988817B - Method for randomly breaking DNA - Google Patents
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Abstract
The invention provides a method for randomly breaking DNA, which comprises the following steps: the DNA molecule is broken in the presence of divalent cations, random DNA double strand binding proteins, non-ionic surfactants, and monovalent salts. The method can effectively improve the efficiency of constructing the second-generation sequencing library.
Description
Technical Field
The present invention relates to a method for randomly breaking DNA.
Background
With the development and maturity of high-throughput sequencing (NGS) technology, the application range in the fields of scientific research, diagnosis and physical examination is continuously expanded. In addition to market expansion, the development of sequencing technology itself slows down, and bottlenecks that restrict the expansion of technology are gradually emerging. Nowadays, bottleneck effect of sample preparation in NGS is more and more obvious, and new reagent and new instrument are emerging continuously to shorten time and improve throughput. At the same time, new methods are being developed to handle smaller sample starting volumes, etc.
At present, there are four main methods for fragmenting chromosome Deoxyribonucleic acid (DNA) molecules, which are respectively: DNA restriction, hydrodynamic shearing, ultrasonic fragmentation, and jet atomization, each of which has advantages and disadvantages. The main reason for fragmenting long-chain DNA (usually referred to as biological chromosomal DNA) is that short DNA fragments are advantageous for rapid hybridization of DNA molecules and highly sensitive target detection, and in addition, fragmentation is an unavoidable process in the construction of NGS libraries. With the continuous and intensive research, researchers focusing on DNA fragmentation technology are increasingly demanding smaller, faster, more efficient and less polluting real-time detection systems, and thus the demand for DNA fragmentation technology is also increasing. The double helix structure of the DNA molecule is a relatively stable structure, two long deoxynucleotide chains are stably connected in parallel through base pairs formed by hydrogen bonds on the inner side, and meanwhile, the stability of the DNA molecule is further improved through the longitudinal interaction force between the base pairs. Currently, the search for new and effective DNA fragmentation techniques is a very research-meaningful but extremely challenging topic.
Disclosure of Invention
A fragmentation method based on hydrodynamic shear method and ultrasonic fracture method is a DNA molecule fragmentation method recommended by Illumina official at the earliest. But the requirements and limitations of corresponding instrument matching become bottlenecks in the popularization and throughput improvement of the method. In addition, the specificity of the DNA molecule, such as the degree of methylation modification and the like, can improve the characteristics of the molecule, and the mechanical method can be used for leading the region to have different fragmentation ratios (probability) with other regions, so that the preference of mechanical disruption is introduced to a certain degree.
In recent years, a new random fragmentation method of non-restriction enzyme treatment is successively appeared in the market of NGS library construction kit, which greatly reduces the dependence on the instrument. However, it also has certain limitations, mainly due to the stability, preference, sensitivity to the initial amount and compatibility of the subsequent library construction steps of the enzyme itself. For example, the drawbacks exhibited by the company Illumina based on the Tn5 transposase are the stability of the enzyme itself, preference, sensitivity to the starting amount; the disadvantage of NEB based on T7 dnas endonuclease is the stability of the enzyme itself, the sensitivity to the initial amount and the compatibility with the subsequent pooling steps; the KAPA system based on Shrimp Dnase has the disadvantage of increasing the requirements for sample purity and for starting amount.
Therefore, in view of the above circumstances, the problem to be solved by the present invention is to use a lower cost non-restriction enzyme method to establish a DNA fragmentation method suitable for common molecular biology laboratories and high throughput NGS library construction laboratories, without the limitation of fragmentation instruments.
The invention mainly relates to the following specific schemes:
1. a method of randomly disrupting DNA, comprising:
the DNA molecule is broken in the presence of divalent cations, random DNA double strand binding proteins, non-ionic surfactants, and monovalent salts.
2. The method according to item 1, wherein,
the divalent cation is one or more than two selected from calcium ion and magnesium ion.
3. The method according to item 1 or 2, wherein,
the random DNA double strand binding protein is sso7d protein.
4. The method according to any one of items 1 to 3, wherein,
the non-ionic surfactant is one or more than two selected from TritonX-100, Tween-20 and Tween-80.
5. The method according to any one of items 1 to 4, wherein,
the metal ions in the monovalent salt are selected from one or more of sodium ions and potassium ions.
6. The method according to any one of items 1 to 5, wherein,
the divalent cation is used at a concentration of 0.01 mM-2 mM, preferably 0.05 mM-1 mM, and more preferably 0.05 mM-0.5 mM.
7. The method according to any one of items 1 to 6, wherein,
the concentration of the random DNA double-strand binding protein is 0 ng/. mu.L to 6 ng/. mu.L, preferably 3 ng/. mu.L to 6 ng/. mu.L, and more preferably 4 ng/. mu.L to 6 ng/. mu.L.
8. The method according to any one of items 1 to 7, wherein,
the nonionic surfactant is used at a concentration of 0.001% to 1%, preferably 0.005% to 0.5%, and more preferably 0.01% to 0.2%.
9. The method according to any one of items 1 to 8, wherein,
the monovalent salt is used in a concentration of 0mM to 500mM, preferably 20mM to 200 mM. More preferably 50 mM-100 mM.
10. The method according to any one of items 1 to 9, wherein,
random DNA fragmentation is performed using a non-restriction endonuclease in the presence of divalent cations, random DNA double strand binding proteins, non-ionic surfactants, and monovalent salts.
11. The method of item 10, wherein,
the non-limiting endonuclease is one or more than two of DNAse I, heat-labile shrimp DNAse, HL-dsDNase and dsDNase.
ADVANTAGEOUS EFFECTS OF INVENTION
By adopting the method of the invention, a high-value DNA breaking instrument is not needed, and efficient and uniform fragmentation reaction can be carried out only by using isothermal bath equipment of a PCR instrument commonly used in molecular biology laboratories, the treatment time is as short as about 10 minutes, and the treatment flux is greatly improved while the cost is saved.
Unlike the random ends generated by the mechanical disruption method in the prior art, the 5 'end of the DNA fragment disrupted by the method is a phosphate group, and the 3' end is a hydroxyl group, but the method in the prior art cannot realize the control of the ends, so that the step of repairing the single-stranded ends in the construction of the NGS library is saved, the excessive fragmentation loss of the highly methylated genome region is reduced, and the efficiency (i.e. the power) of the library construction is improved.
The method disclosed by the invention is insensitive to the structure of the purified DNA molecule, can be used for processing samples of different species and tissue sources in a high-throughput manner under the same operation condition, efficiently obtains products with a narrow molecular weight range without preference, and completely meets the requirement of constructing a downstream NGS library. The purity standard of sample admission is reduced.
After the fragmentation reaction, the downstream NGS library building operation can be directly carried out while the fragmentation reaction is carried out, the operation of system replacement or DNA purification and separation is not needed to be added, and the reaction is terminated only by raising the temperature for heat inactivation. The fluency of the whole library construction process is further improved.
Drawings
Figure 1 shows that adjusting ion concentration reduces site preference.
Figure 2 shows that the addition of random DNA double strand binding protein reduces site bias.
FIG. 3 shows the average moisture content of the target region after addition of random DNA double strand binding protein.
Figure 4 shows the results of the nonionic surfactant to improve breaking uniformity.
FIG. 5 shows the results of increasing monovalent salt concentration to improve breaking stability.
FIG. 6 shows the library concentrations resulting from constructing libraries using the disruption method of the present invention.
FIG. 7 shows the number of undetected sites for library construction using the disruption method of the present invention.
FIG. 8 shows repeated useless predicate numbers for library construction using the disruption method of the present invention.
FIG. 9 shows coverage of captured target regions for library construction using the disruption method of the present invention.
FIG. 10 shows aligned genomic read outs from libraries constructed using the disruption method of the invention.
FIG. 11 contains a caliper peak plot of the library constructed after disruption with another endonuclease, HL-dsDNase.
FIG. 12 shows calipe peak plots for library construction using another monovalent salt KCL disruption method.
FIG. 13DnaseI library caliper peak plot.
FIG. 14KAPA library caliper peak plot.
Detailed Description
The random fragmentation method of non-restriction endonuclease treatment needs to overcome the stability, preference, sensitivity to initial amount and compatibility of subsequent library building steps of the enzyme. In the present invention, a wide source of non-restriction enzymes is selected and used, so that the randomness problem of the distribution of the sites is not considered, and after all, the restriction enzymes are usually used on DNA from a wide variety of sources, and the preference sites of the restriction sites of different species are very different. In addition, T7 is not used
Endonucleases this thermodynamically favored enzyme with single strand as substrate contributes to the preference for GC content in the DNA molecule region.
In the present invention, non-limiting enzymes having single-and double-stranded activities are selected and used, and examples thereof include, but are not limited to: DNAse I, thermolabile shrimp DNAse (thermolabile shrimp DNAse), HL-dsDNase, etc. This type of DNase shows a preference for cleavage sites under traditional buffer systems. The invention overcomes the problems of stability, preference, sensitivity to initial amount, compatibility of subsequent library building steps and the like of the enzyme by adjusting a buffer system and adding an additive.
DNase I (Deoxyribonuclease I), the Chinese name "Deoxyribonuclease I", is an endonuclease that digests single-or double-stranded DNA to produce either single-or double-stranded oligodeoxynucleotides. The 5 'end of the product after the DNase I hydrolyzes the single-chain or double-chain DNA is a phosphate group, and the 3' end is a hydroxyl group. DNase I activity is dependent on calcium ions and can be activated by magnesium ions or divalent manganese ions. In the presence of magnesium ions, DNase I can randomly cut any site of double-stranded DNA; reduction of Ca2+The ratio of activated DNase I to DNA molecule sites, whereby site preference may be reduced; another means for reducing preference is to add random DNA double-strand binding protein to cover the DNA molecule at a certain saturation level, and DNAse I can only enter the uncovered region without preference to act.
Thermolabile shrimp DNAse (thermolabile shrimp DNAse) is a DNAse extracted from Antarctic deep sea shrimp.
dsDNase is a novel double-stranded specific DNase, a shrimp DNase designed for rapid and safe removal of genomic DNA from RNA samples. It is a nuclease that cleaves phosphodiester bonds in DNA, producing oligonucleotides, 5 '-phosphates and 3' -hyds.
HL-dsDNase (Heat-laboratory Double Stranded DNase) is a genetically engineered product of dsDNase, and can be completely inactivated rapidly at moderate temperature (55 ℃, pH greater than 8.0). Is ideally suited for use in the removal of DNA contamination from RNA (possibly in the presence of magnesium ions).
The activity of the above-mentioned non-restriction enzymes is greatly influenced due to the molecular structure openness of DNA. In the process of cutting the DNA molecule by enzyme, the configuration is changed sharply, and some regions with stable tertiary structures are easy to remain, which influences the entry and exit of the non-restriction enzyme molecule, and further shields the capability of cutting the whole DNA molecule uniformly. Therefore, in order to facilitate rapid entry and exit of the enzyme into and out of these areas, the present invention uses a nonionic surfactant. In addition, DNA samples obtained by different extraction methods have different degrees of protein, salt and carbohydrate residues, although they are avoided as much as possible. This also affects the conformation and surface opening of the DNA molecule to a certain extent, and the use of surfactants can promote the rapid dissociation of proteins from the DNA molecule without hindering the action of the above-mentioned non-restriction enzymes. The salt concentration of more than 50-100mM has obvious inhibition effect on the non-restriction enzyme, and is mainly derived from the increase of tertiary structure. However, the influence of low-concentration salt on the activity is unstable, the invention actively increases the concentration of metal ions in monovalent salt in a buffer system, and the unstable cutting caused by buffering the low-concentration salt by a method of increasing the enzyme input amount.
The Sso7d protein is a double-stranded DNA sequence non-specifically binding protein.
The present invention requires compatibility with the subsequent NGS library construction steps, controls the degree of reaction by time, and requires a means of interrupting fragmentation. Heat inactivation of the fragmentation enzyme is the first means of the least number of steps, and the reaction for the first step of library construction is usually a polymerase reaction environment, containing the substrates necessary for the polymerase reactions. For example, the above-mentioned non-limiting endonuclease is just one enzyme that can be heat-inactivated and that can function normally under various polymerase systems.
Examples
The present invention will be described in detail below with reference to examples. In the following examples, each material used may be obtained commercially, unless otherwise specified, and the method used is a conventional method in the art, unless otherwise specified. Unless otherwise specified, percentages indicate weight percentages.
The ingredients of the buffer component in the reactions referred to in the examples are first listed
1 Blue Buffer (purchased from enzymics):
10mM Tris-Hcl
10mM MgCl2
50mM Nacl
1mM DTT
PH7.9 at 25℃
0.1 XDnaseI Buffer (purchased from NEB):
1mM Tris-HCl
0.25mMMgCl2
0.05mM CaCl2
PH7.6 at 25℃
example 1 adjusting ion concentration to reduce site preference
Interrupting the reaction operation process
Taking 200ng of human saliva genome DNA as an initial sample, adding 0.02-8 XDnaseI Buffer, 1 Xblue Buffer and 2.5mU DNaseI into a reaction system, and supplementing ddH2O to 10. mu.L. The PCR instrument was set at 37 ℃ for 60min and the reaction was stopped by adding 5mM EDTA. After the reaction is finished, the size of the broken fragment is detected by 2% agarose gel electrophoresis, the voltage is set to be 120V by an electrophoresis apparatus, and the time is 30 min. FIG. 1 shows the results of the above electrophoresis, and the upper part of FIG. 1 shows the concentrations of calcium ions in different lanes, and it can be known from the results shown in FIG. 1 that 0.05mM Ca2+The need for disruption can be met and the loss of DNA from excessive disruption is reduced.
Example 2 addition of random DNA double-stranded binding protein to reduce site bias
Interrupting the reaction and constructing the library
Taking 400ng of human salivary genome DNA extracted by a Tiangen extraction kit and a Ranunculus sieboldii extraction kit as initial samples, adding 0.1 XDNaseI Buffer, 1 Xblue Buffer, 0-120ng sso7d, 5mU DNaseI into a reaction system, and supplementing ddH2O to 20. mu.L. The PCR instrument was set at 37 ℃ for 60min and the reaction was stopped by adding 5mM EDTA. After the reaction is finished, detecting the size of the broken fragment by using 2% agarose gel electrophoresis, setting the voltage of an electrophoresis apparatus to be 120V, and setting the time to be 30 min.
Taking 100ng of the above interruption product as an initial sample, and performing library preparation according to the traditional small fragment library building process, wherein the PCR reaction program is set as: 94 ℃ for 2 min; repeating the cycle of 94 ℃ for 15s, 62 ℃ for 30s and 72 ℃ for 30s for 10 times; 10min at 72 ℃; maintained at 4 ℃. And (3) after the amplification is finished, purifying PCR products: the DNA in the reaction system was recovered and purified using 0.9 × Ampure Beads, and eluted with 25 μ L EB. The small fragment library construction is complete. Library detection: library yields were measured using an Agilent 2100 Bioanalyzer and quantified using qPCR. The results of the disruption reactions are shown in FIG. 2. Data alignment results are shown in fig. 3, which shows the mean depth of the target region for three samples (3 samples are independent 3 samples, 3 biological replicates). From the results of FIG. 3, it was found that 120ng to 0ng, the depth of detection at the same site was decreased.
Example 3 use of nonionic surfactant to improve breaking uniformity
Interrupting the reaction operation process
Taking 200ng of human saliva genome DNA of 5 different sources as a starting sample, adding 0.1 XDnaseI Buffer, 1 XBlue Buffer, 0.1% TritonX-100 or 0.01% Tween-20, 2.5mU DNaseI into a reaction system, and supplementing ddH2O to 10. mu.L. The PCR instrument was set at 37 ℃ for 60min and the reaction was stopped by adding 5mM EDTA. After the reaction is finished, detecting the size of the broken fragment by using 2% agarose gel electrophoresis, setting the voltage of an electrophoresis apparatus to be 120V, and setting the time to be 30 min. The results of the interrupting reactions are shown in FIG. 4. from the results in FIG. 4, it can be seen that the interrupting homogeneity is improved by adding 0.1% TritonX-100 or 0.01% Tween-20。
Example 4 increasing the concentration of Metal ions in monovalent salts improves breaking stability
Interrupting the reaction operation process
400ng of human saliva genome DNA was used as a starting sample, and 0.1 XDnaseI Buffer, 1 Xblue Buffer, 0-50mM NaCl, 0.1% TritonX-100, 120ng sso7d, 10mU DNaseI, and ddH were added to the reaction system2O to 40. mu.L. The PCR instrument was set to 37 ℃ for 10min, and the reaction was terminated by adding 5mM EDTA. After the reaction is finished, detecting the size of the broken fragment by using 2% agarose gel electrophoresis, setting the voltage of an electrophoresis apparatus to be 120V, and setting the time to be 30 min. The results of the reaction are shown in FIG. 5, and according to the experimental results, 50mM Na was used+The stability of the breaking reaction is improved.
Example 5
And (3) interrupting the reaction operation flow:
taking 200ng of saliva sample genome DNA extracted by 12 meiji kits as an initial sample, carrying out DnaseI enzyme digestion interruption and ultrasonic interruption on each sample in parallel, setting an ultrasonic interruption system as 400ng of DNA and 75 mu L of the DNA, and interrupting conditions: 30 seconds on/30 seconds off, 17 cycles. Adding 0.1 XDnaseI Buffer, 1 Xblue Buffer, 50mM NaCl, 0.1% TritonX-100, 120ng sso7d, 10mU DNaseI and complement ddH into a DnaseI enzyme digestion interruption reaction system2O to 40. mu.L, and the PCR program was set to 37 ℃ for 10min and 75 ℃ for 20 min. The method comprises the following steps of directly taking 100ng of enzyme digestion breaking products and 200ng of ultrasonic breaking products without purification, building a library according to the traditional small fragment library building process, carrying out quantitative hybridization by using the Qubit, carrying out sequencing data analysis, and carrying out a sequencing platform: nextseq550, sequencing type: PE150, data size requires 0.4G clean data.
Experimental results the results of the experiments are described below, and fig. 6 shows the library concentrations obtained from constructing the libraries according to the method described above. According to the library concentration in FIG. 6, 200ng of the cleavage product by ultrasonic and 100ng of the cleavage product by enzyme are shown, and the efficiency of cleavage is obviously higher than that of cleavage by ultrasonic, which indirectly indicates the cleavage product. According to the sequencing results (FIG. 7-FIG. 10), enzyme cutting interruption of each sequencing index is superior to ultrasonic interruption. The effect obtained by the method of the invention is significantly better than the ultrasound interruption of the prior art.
Example 6
(1) And (3) changing the type of the endonuclease:
interrupting the reaction operation process
Taking 3 200ng of human saliva genome DNA as a starting sample, adding 0.1 XDnaseI Buffer, 1 XBlue Buffer, 50mM NaCl, 0.1% TritonX-100, 120ng sso7d, 20mU HL-dsDnase and supplement ddH into a reaction system2O to 40. mu.L, and the PCR program was set to 37 ℃ for 10min and 75 ℃ for 20 min. Library construction was performed according to the conventional small fragment library construction procedure, and library peak patterns were detected using Caliper, as shown in fig. 11.
Under the enzyme cutting system, another endonuclease HL-dsDNase (Heat-laboratory Double Stranded DNase) is used for cutting, and the cutting effect is stable.
(2) Change of monovalent salt:
interrupting the reaction operation process
Taking 3 200ng of human saliva genome DNA as a starting sample, adding 0.1 XDnaseI Buffer, 1 XBlue Buffer, 50mM KCl, 0.1% TritonX-100, 120ng sso7d, 10mU DnaseI and complement ddH into a reaction system2O to 40. mu.L, and the PCR program was set to 37 ℃ for 10min and 75 ℃ for 20 min. Library construction was performed according to the conventional small fragment library construction procedure, and library peak patterns were detected using Caliper, as shown in fig. 12. The KCl is used for interruption under the interruption system, and the interruption effect is stable.
Comparative example 1
And (3) interrupting the reaction operation flow:
taking 100ng of NA12878 standard as an initial sample, performing DnaseI library interruption and KAPA kit library interruption in parallel, and setting 3 technical repeats. Adding 0.1 XDnaseI Buffer, 1 Xblue Buffer, 50mM NaCl, 0.1% TritonX-100, 120ng sso7d, 10mU DNaseI and complement ddH into a DnaseI enzyme digestion interruption reaction system2O to 40. mu.L, and the PCR program was set to 37 ℃ for 10min and 75 ℃ for 20 min. The impure product is directly taken and digested by 100ng, and the library is built according to the traditional small fragment library building process, and the result is shown in FIG. 13. The KAPA kit system was subjected to library construction according to kit instructions, and the results are shown in FIG. 14.
The experimental results show that the stability of the size of the DnaseI disrupted fragment is better than that of the KAPA kit, and the concentration of the library is higher than that of the KAPA kit.
Claims (3)
1. A method of randomly disrupting DNA, comprising:
cleaving a DNA molecule with a non-restriction enzyme in the presence of a divalent cation, a random DNA double strand binding protein, a non-ionic surfactant, and a monovalent salt, wherein,
the divalent cation is one or two selected from calcium ions and magnesium ions, the use concentration of the divalent cation is 0.04 mM-0.1 mM,
the random DNA double-strand binding protein is sso7d protein, the use concentration of the random DNA double-strand binding protein is 3 ng/muL-6 ng/muL,
the non-ionic surfactant is one or more than two selected from TritonX-100, Tween-20 and Tween-80, the use concentration of the non-ionic surfactant is 0.01-0.2%,
the metal ions in the monovalent salt are one or two selected from sodium ions and potassium ions, the use concentration of the monovalent salt is 20 mM-200 mM,
the non-limiting endonuclease is one or two selected from DNAse I and HL-dsDNase.
2. The method of claim 1, wherein,
the concentration of the random DNA double-strand binding protein is 4 ng/mu L-6 ng/mu L.
3. The method of claim 1, wherein,
the monovalent salt is used at a concentration of 50mM to 100 mM.
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| Title |
|---|
| Kentaro Miyazaki.Random DNA fragmentation with endonuclease V:application to DNA shuf¯ing.《Nucleic Acids Research》.2002,全文. * |
| Random DNA fragmentation aFFPE samplesllows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical;Galen Hostetter et al.;《Nucleic Acids Research》;20091029;全文 * |
| Using archaeal histones for precise DNA fragmentation;E. Azzoni et al.;《Protein Engineering Design and Selection》;20070730;全文 * |
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