HK1237002B - Primer for nucleic acid random fragmentation and nucleic acid random fragmentation method - Google Patents

Description

A primer for random nucleic acid fragmentation and a method for random nucleic acid fragmentation

Technical Field

The present application relates to the field of nucleic acid fragmentation processing, and in particular to a primer for random nucleic acid fragmentation, and a method for random nucleic acid fragmentation based on the primer.

Background Art

Since Roche invented pyrosequencing, pioneering next-generation sequencing (NGS), it has experienced rapid growth. However, with the development of high-throughput sequencing, high-throughput and low-cost sample preparation has become a key consideration in the sequencing field. Various sample processing methods and automated devices have been developed, primarily for sample fragmentation, nucleic acid end-processing, and adapter ligation.

Sample fragmentation is primarily achieved through physical methods, such as ultrasonic shearing, or enzymatic methods, which involve non-specific endonuclease treatment. The primary physical method is the Covaris, based on patented Adaptive Focused Acoustic (AFA) technology. Under isothermal conditions, this method geometrically focuses acoustic energy, with a wavelength of 1 mm, onto the sample via a spherical solid-state ultrasonic transducer operating at >400 kHz. This method ensures the integrity of nucleic acid samples and achieves high recovery rates. Covaris instruments include the economical M series, the single-tube, full-power S series, and the higher-throughput E and L series. While fragmentation based on physical methods offers high randomness, throughput relies on a large number of Covaris fragmentation instruments, requiring subsequent end-treatment, adapter addition, PCR, and various purification steps. Enzymatic methods include NEB Next dsDNAFragmentase, launched by NEB. This reagent first creates random nick sites in double-stranded DNA, then a second enzyme recognizes the nick sites and cleaves the complementary DNA strand, achieving fragmentation. This reagent can be used for genomic DNA, whole-genome amplification products, and PCR products, and has good randomness. However, it can produce some artificial short insertions and deletions, and inevitably requires subsequent end-treatment, adapter addition, PCR, and corresponding purification operations. Transposase fragmentation kits, led by Epicentra's Nextera kit, utilize transposase to simultaneously fragment DNA and add adapters, thereby reducing sample processing time.

From the perspective of operational simplicity, transposase fragmentation undoubtedly surpasses other methods in terms of throughput and operational ease. However, this fragmentation method also has its own drawbacks: transposase relies on a specific 19-bp Me sequence for transposition. Therefore, while the transposase can add different adapter sequences to the 5' and 3' ends of the target sequence by embedding two completely different adapter sequences, both adapters must contain a specific Me sequence. This results in fragments with symmetrical Me sequences at either end. Furthermore, due to the specific action of the transposase, a 9-nt base gap is missing between the target sequence or the fragmented fragment and the Me sequence. Identical Me sequences at both ends of the adjacent target sequence can impact downstream applications, such as ligation-based next-generation sequencing, where the Me sequences on either side of the same strand are complementary, which can easily cause annealing within the single-stranded molecule and hinder the binding of the anchor primer.

A related patent application, CN 102703426 A, once proposed a solution, which is to subject the interrupted sequence to a specific endonuclease digestion to remove the 9nt sequence and Me sequence. This method simply takes advantage of the transposase interruption to randomly interrupt the nucleic acid sequence, but introduces the disadvantage that subsequent linkers need to be added separately. The steps are cumbersome and not easy to apply to higher throughput.

Summary of the Invention

The purpose of this application is to provide a new primer for random nucleic acid fragmentation, and on this basis, to provide a new DNA double random primer fragmentation method, that is, a method for random nucleic acid fragmentation.

In order to achieve the above objectives, this application adopts the following technical solutions:

On the one hand, the present application discloses a primer for random nucleic acid fragmentation, which consists of several upstream random primers and several downstream random primers; the sequence composition of the upstream random primer is: 5'-X-Y-3'; the sequence composition of the downstream random primer is: 5'-P-Y'-X'-close-3'; wherein Y and Y' are random sequences, X is the entire or partial sequence of the 5' end adapter of the sequencing platform, X' is the entire or partial sequence of the 3' end adapter of the sequencing platform, P is a phosphorylation modification, and close is a blocking modification used to prevent the formation of a 3-5 phosphodiester bond.

It should be noted that the upstream random primer and the downstream random primer of the present application are different from conventional PCR primers. According to the design, the upstream random primer and the downstream random primer are hybridized and bound to the same template chain. Moreover, during the extension stage, only the space between the upstream random primer and the downstream random primer closest to each other is filled by the upstream random primer extending toward the 3' end. Since the 5' end of the downstream random primer is phosphorylated, the 3' end of the extended sequence of the upstream random primer can be connected to the 5' end of the downstream random primer, that is, the upstream random primer and its extended sequence are connected to the downstream random primer into a section. Since the upstream random primer and the downstream random primer are both randomly hybridized to the template, random fragmentation of the DNA sample can be achieved. Unlike conventional random primer amplification of DNA samples, in the primers of the present application, only the upstream random primer is extended, and the extension stops immediately when it reaches the downstream random primer. Unlike random primer amplification of DNA samples, in the second round of amplification, the amplified chain of one upstream primer may correspond to multiple downstream primers, thereby amplifying a series of diffuse bands, affecting the accuracy of the results.

It should also be noted that the two random sequences Y and Y' in this application, like those of conventional random primers, consist of 3-12 bases, preferably 5-9 bases. Sequences X and X' are the adapter sequences of the sequencing platform, preparing for subsequent sequencing. X represents the entire or partial sequence of the 5' adapter of the sequencing platform because, after random fragmentation of the DNA sample, a pair of universal primers is required to amplify the signal of the purified random DNA fragments, i.e., PCR amplification. When the primers for this PCR amplification use the random DNA fragments as templates for amplification, the entire sequence of the 5' adapter can be complemented through primer design. Therefore, the X sequence in the upstream random primer can be the entire or partial sequence of the 5' adapter of the sequencing platform. Based on the same considerations, X' represents the entire or partial sequence of the 3' adapter of the sequencing platform.

Preferably, the blocking modification is a dideoxy modification. It should be noted that the role of the blocking modification is to prevent the 3' end extension of the downstream random primer and to prevent the connection between the downstream random primers; therefore, in theory, any blocking modification that can prevent the formation of 3-5 phosphodiester bonds can be used in this application. After comprehensive consideration of various aspects, this application confirms that the dideoxy modification can both play a blocking role and not affect the hybridization of random primers.

Preferably, the 5' end of the X sequence of the upstream random primer further includes 2-6 protection bases; the 3' end of the X' sequence of the downstream random primer further includes 2-6 protection bases, and the blocking modification is on one of the terminal protection bases.

It should be noted that the role of the protective base is to make the sequence more stable. It is understandable that, under lower requirements, the protective base may not be designed.

Preferably, several spacer bases are included between the X and Y sequences of the upstream random primers; and several spacer bases are included between the Y' and X' sequences of the downstream random primers. It should be noted that spacer bases are not required. Their function is also to ensure sequence stability. However, spacer bases will be amplified during the subsequent PCR amplification after purification, that is, they will create gaps between the adapter and the randomly interrupted DNA fragments. Therefore, unless there are special requirements, it is best not to add spacer bases.

In one implementation of the present application, the X sequence of the upstream random primer has the sequence shown in Seq ID No. 1, and the X' sequence of the downstream primer has the sequence shown in Seq ID No. 2;

Seq ID No.1: 5’-GACCGCTTGGCCTCCGACT-3’

Seq ID No. 2: 5’-GTCTCCAGTCGAAGCCCGA-3’.

Another aspect of the present application discloses a method for random nucleic acid fragmentation, comprising using the primers of the present application to perform double random anchoring on a DNA sample, specifically comprising hybridizing an upstream random primer and a downstream random primer to a denatured DNA sample, extending the upstream random primer toward its 3' end between the most adjacent upstream random primer and the downstream random primer through the action of a DNA polymerase, thereby filling the sequence between the upstream random primer and the downstream random primer, and then ligating the 3' end of the extended sequence of the upstream random primer to the 5' end of the downstream random primer under the action of a DNA ligase, that is, connecting the upstream random primer and its extended sequence to the downstream random primer into a segment, thereby achieving double random fragmentation of the DNA sample through random hybridization of the upstream random primer and the downstream random primer.

It should be noted that the method of the present application is to add a certain amount of upstream random primers and downstream random primers and a denatured DNA sample to a reaction solution containing DNA polymerase, DNA ligase and dNTPs to achieve extension and ligation reactions. In fact, the hybridization of the upstream random primer and the downstream random primer in the present application, and the extension of the upstream random primer, are the same as the principles of conventional PCR. The random primers first hybridize to the denatured DNA sample, and then under the action of DNA polymerase, the 3' end of the upstream random primer begins to extend. Since only the 5' end of the downstream random primer is phosphorylated, only the most adjacent upstream random primer and downstream random primer can be connected into a segment by DNA ligase after extension, thereby ensuring the accuracy of random interruption and avoiding the introduction of erroneous structures.

Preferably, in the process of hybridizing the upstream random primer and the downstream random primer to the denatured DNA sample, the total amount of the upstream random primer and the downstream random primer is R×n picomoles, wherein 2.7≤R≤750, n=1.515×(m÷L), m is the weight of the DNA sample in nanograms, L is the expected length of the DNA fragment after shearing, and n is the theoretical amount of the upstream random primer and the downstream random primer required to break the DNA sample into fragments of L length, in picomoles.

It should be noted that the amount of upstream random primers and downstream random primers used is related to the degree of fragmentation required. It can be understood that the greater the amount of upstream random primers and downstream random primers used, the denser the random primers hybridized to the DNA molecule chain, the smaller the distance between the most adjacent upstream random primers and downstream random primers, and thus the smaller the connected fragments, that is, the smaller the obtained DNA fragments, that is, the higher the degree of fragmentation; conversely, the obtained randomly interrupted DNA fragments are larger; the present application has deduced that the theoretical amount of primers required to fragment a DNA sample into fragments of length L is n, and based on a large number of experimental analyses, it has been concluded that, depending on the requirements of different fragmentation lengths L, the total amount of upstream random primers and downstream random primers used is actually R times the theoretical amount n, therefore, R×n picomoles, the larger R is, the smaller the fragmented fragment L, and L is the number of base pairs of the fragments after fragmentation, in bp.

Preferably, the molar ratio of the upstream random primer to the downstream random primer is upstream random primer: downstream random primer = 1 to 3:1, preferably 2:1, and preferably R=20.

Another aspect of the present application discloses a method for constructing a nucleic acid library, comprising randomly fragmenting a DNA sample using the nucleic acid random fragmentation method of the present application, and then using a pair of universal primers to PCR amplify the double-randomly interrupted DNA fragments, thereby obtaining a nucleic acid library enriched in random fragments; the universal primers are composed of a forward primer and a reverse primer, the 3' end of the forward primer has all or part of the sequence of the 5' end adapter of the upstream random primer sequencing platform, and the 3' end of the reverse primer has all or part of the reverse complementary sequence of the 3' end adapter of the downstream random primer sequencing platform.

It should be noted that the forward primer and reverse primer mentioned in the primer set of the present application, i.e., conventional PCR amplification primers, are designed for randomly fragmented DNA fragments, and PCR amplification is performed using the randomly fragmented DNA fragments as templates to achieve signal amplification of the randomly fragmented DNA fragments; therefore, the 3' end of the forward primer has all or part of the sequence of the 5' end adapter of the sequencing platform, and the 3' end of the reverse primer has all or part of the reverse complementary sequence of the 3' end adapter of the sequencing platform. Since the upstream random primer and the downstream random primer contain all or part of the adapter sequence, and the forward primer and the reverse primer also contain part or all of the sequence, it is sufficient that the PCR amplification product contains the entire adapter sequence. For example, using only the upstream random primer and the forward primer as an example, when the X sequence of the upstream random primer is the entire sequence of the 5' adapter on the sequencing platform, the 3' end of the forward primer can be the entire sequence of the 5' adapter or a partial sequence of the 5' end of the 5' adapter, as long as it can hybridize with the X sequence of the upstream random primer to amplify the complete 5' adapter sequence. Similarly, when the X sequence of the upstream random primer is a partial sequence of the 5' adapter on the sequencing platform, the 5' adapter sequence can be complemented by the forward primer. In this case, the 3' end of the forward primer can be the entire sequence of the 5' adapter or a complementary sequence as well as a sequence that partially hybridizes with the 5' adapter sequence in the X sequence of the upstream random primer, thereby amplifying the complete 5' adapter. Therefore, the 3' end of the forward primer can be all or part of the sequence of the 5' end adapter of the sequencing platform; similarly, the 3' end of the reverse primer has all or part of the reverse complementary sequence of the 3' end adapter of the sequencing platform.

It should also be noted that although the primers of the present application can well ensure the random interruption of DNA samples and will not produce diffuse fragment interference, since no actual PCR amplification is performed when connecting the upstream random primer and the downstream random primer, only the upstream random primer is extended. Therefore, the copy number of the random DNA fragments is limited. Therefore, when sequencing or library construction, it is necessary to use forward primers and reverse primers to PCR amplify the randomly interrupted DNA fragments, and the forward primers and reverse primers are designed based on the adapter of the sequencing platform. In other words, all random DNA fragments actually contain part or all of the sequence of the sequencing platform adapter. After the adapter sequence of the sequencing platform is determined, the forward primer and reverse primer will amplify all DNA fragments. Therefore, they are called universal primers. It should also be noted that usually after connecting to randomly interrupted DNA fragments, the connected fragments need to be purified and then PCR amplified. The method for purifying the connected randomly interrupted DNA fragments can refer to conventional DNA purification methods. The present application preferably uses a magnetic bead method to separate and purify the DNA fragments.

Preferably, the 5' ends of the forward primer and the reverse primer respectively have an adapter sequence of the second sequencing platform.

It should be noted that the 5’ ends of the forward primer and the reverse primer respectively have the adapter sequence of the second sequencing platform, which is not necessary; it can be understood that if sequencing is only required on one sequencing platform, only the 5’ end adapter and 3’ end adapter of one sequencing platform are required, and the adapter of the second sequencing platform is to facilitate subsequent sequencing needs.

In one implementation of the present application, the forward primer contains the sequence shown in Seq ID No. 1, and the reverse primer contains the sequence shown in Seq ID No. 3;

Seq ID No. 3: 5’-TCGGGCTTCGACTGGAGAC-3’.

Due to the adoption of the above technical solution, the beneficial effects of this application are:

The present application primers and the method for random fragmentation of nucleic acids based on the primers are applicable to various DNA samples, including random fragmentation of cDNA sequences. The upstream and downstream double random primers can cover almost the complete sequence of the target sequence and achieve a higher coverage uniformity, thereby providing a better basis for subsequent molecular biology operations and information mining. In addition, the random fragmentation method of the present application only requires several conventional tool enzymes and PCR nucleic acid amplification equipment to complete the sample processing of the whole process, thereby making it possible for small and medium-sized scientific research institutions, colleges and universities, and downstream application fields to independently carry out high-throughput library preparation. At the same time, the present invention only needs to denature the target sequence, and then the random anchoring and polymerization ligation reaction of the random primers can be used for subsequent amplification, so as to have the advantages of simplicity and speed, get rid of the dependence on large-scale high-end instruments and equipment or expensive test kits, and its simple and easy-to-operate characteristics greatly reduce the professional skills requirements for technical personnel, greatly widening the application field of large-scale high-throughput sequencing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a technical breakdown diagram of the random DNA fragmentation method according to an embodiment of the present invention;

Figure 2 is a diagram showing the electrophoresis results of the optimized conditions in the examples of the present application;

Figure 3 is a diagram showing the electrophoresis results of the optimized conditions in the examples of this application.

DETAILED DESCRIPTION

The primers designed in this application bind both the upstream random primer and the downstream random primer to the same template strand, rather than to two complementary template strands. Their purpose is not to perform PCR amplification, but rather, as shown in Figure 1, to fill the gap between adjacent upstream random primers and downstream random primers by extending the upstream random primer, which are then ligated into complete fragments using DNA ligase. Because both the upstream and downstream primers are random, double random fragmentation of the DNA sample can be achieved. Furthermore, because the upstream random primer stops extending after reaching the downstream random primer, this avoids the problem of random primers amplifying the DNA sample during the second round of PCR amplification, where a single upstream primer amplification strand corresponds to multiple downstream primers, resulting in diffuse fragment bands and compromising subsequent operations.

The method for random nucleic acid fragmentation based on the primers of the present application requires a short time and is almost entirely completed on a PCR instrument. It can achieve the fastest sample preparation and can realize automated operation. Compared with other methods, it can effectively reduce human error and reduce systematic errors in sample processing. At the same time, it also gets rid of the dependence on large-scale high-end instruments and equipment or expensive test kits.

It should be noted that in the method of random fragmentation of nucleic acids in the present application, upstream random primers and downstream random primers are hybridized to denatured DNA samples, wherein the denaturation of the DNA sample can be carried out by high temperature treatment or chemical reagent denaturation method. The time of high temperature treatment is inversely proportional to the temperature. The higher the temperature, the shorter the treatment time. The suitable denaturation temperature is 98-95°C, and the treatment time is 1-5 minutes. In one implementation scheme of the present application, 95°C is selected for reaction for 5 minutes. Commonly used denaturing reagents for chemical reagent denaturation method include KOH, NaOH, EDTA, etc., which are not specifically limited in this application. After the chemical reagent is denatured, the annealing reaction needs to neutralize the alkali ion concentration of the reaction system to keep the reaction system in a neutral and suitable salt ion environment. The neutralization buffer can be various low-concentration acid buffers, and only the alkaline solution treatment group is treated with the neutralization buffer, such as a combination buffer of HCl and Tris-HCl.

Can make extension product fragment size meet sequencing platform needs by the molar ratio between regulation and control upstream random primer, downstream random primer and template, do not do specific restriction at this.In the application, the number of random sequence can have and does not need design scheme in upstream random primer and downstream random primer, comprise 5 random bases, 6 random bases, 7 random bases, 8 random bases etc. to guarantee that primer is attached to the different positions of target sequence, and this is identical with conventional random primer, do not do specific restriction at this.Simultaneously for preventing 5 ' random primer and 3 ' random primer from introducing wrong structure in subsequent reaction, 5 ' end design of upstream random primer is modified without phosphorylation, and 5 ' end of downstream random primer has phosphorylation modification, and 3 ' end of downstream random primer carries out blocking modification, to avoid 3-5 phosphodiester bond to form, preferably dideoxy modification.In addition, downstream random primer and upstream random primer can also comprise the follow-up joint sequence that is applicable to different sequencing platforms.

In addition, the DNA polymerase can be a conventional DNA polymerase, and the ligase after extension is also a conventional DNA ligase, which is not specifically limited here. After the connection is completed, in one implementation of the present application, the single-stranded DNA is purified using a single-stranded DNA magnetic bead selection method with a magnetic bead concentration of 1.0 times. The PCR process is amplified using single-stranded DNA as a template to achieve signal amplification of randomly broken DNA fragments.

In the present application, the amount of upstream random primers and downstream random primers directly affects the length of the DNA fragments after fragmentation. Therefore, in the preferred embodiment of the present application, the upstream random primers and downstream random primers are added to the DNA sample according to a total amount of R×n picomoles, 2.7≤R≤750, n=1.515×(m÷L), m is the weight of the DNA sample in nanograms, and L is the expected length of the DNA fragments after fragmentation; the above formula n=1.515×(m÷L) is theoretically derived by the inventors of the present application, and the value range of R is based on different fragment size requirements after fragmentation and a large number of experimental analyses. The larger the R value, the smaller the fragments after fragmentation. The derivation process of n=1.515×(m÷L) is as follows:

Taking the 3G human genome as an example, it contains 3×10 ⁹ base pairs, and its mass molar concentration is approximately M=(3×10 ⁹ ×660) g/mol=(3×10 ⁶ ×660) ng/pmol=1.98×10 ⁹ ng/pmol,

The number of moles of genomic DNA with a mass of m (ng) is n ₁ =m÷M=m/(3×10 ⁶ ×660×10 ¹² )mol.

The number of molecules of genomic DNA with a mass of m (ng) is N ₁ =n ₁ ×Na=m÷M×Na,

The theoretical number of random primer molecules required to break a molecule to a length of L (bp) is n ₂ = (3×10 ⁹ ÷ L)×N ₁

The theoretical number of moles of random primers required to break a molecule to a length of L (bp) is n = n ₂ ÷ Na

Therefore, n = n ₂ ÷ Na = (3 × 10 ⁹ ÷ L) × N ₁ ÷ Na = (3 × 10 ⁹ ÷ L) × (m ÷ M × Na) ÷ Na

=3×10 ⁹ ×m÷L÷M=(3×10 ⁹ ÷1.98×10 ⁹ )×m÷L=1.515×m÷L pmol

Where Na is Avogadro's constant, Na=6.02×10 ²³

That is, theoretically, to break m nanograms of the 3G human genome into L-length fragments, n pmol of upstream and downstream random primers are needed. However, based on a large number of experimental analyses, in order to obtain L-length fragments, the total amount of upstream and downstream random primers that actually needs to be added is R times n pmol, that is, R × n picomoles. The smaller L, the larger R.

It should be noted that, according to the derived formula, the theoretical amount n of the upstream random primers and the downstream random primers has no direct relationship with the actual length of the DNA sample in theory. The direct factors affecting n are the length L of the fragments to be obtained and the total weight of the DNA sample. Therefore, the formula for the theoretical amount n derived in this application using the 3G human genome as an example is not limited to the 3G human genome. In other words, the nucleic acid random fragmentation method and library construction method of this application have wide applicability and can be used to process any DNA sample, including cDNA.

It should also be noted that the theoretical dosage n in this application is derived based on double-stranded DNA. Typically, the DNA samples obtained in the experiment are also double-stranded DNA. Therefore, the derivation and limitation of the dosage of upstream and downstream random primers in this application are widely applicable. For some special single-stranded DNA samples, it can be understood that it is sufficient to replace the corresponding mass molar concentration M, and the rest remain the same; the mass molar concentration of single-stranded DNA is approximately M = Y × 324.5 g/mol, where Y is the length of the single-stranded DNA. Since the samples typically obtained are double-stranded DNA, this application does not specifically limit the case of single-stranded DNA.

The present invention is further described in detail below through specific examples and drawings. The following examples are only used to further illustrate the present invention and should not be construed as limiting the present invention.

Example

1. Primer Design

In this example, we designed and ordered upstream random primers and downstream random primers containing 8 random sequence sites. The sequences are as follows:

Upstream random primer: 5'-GAC GACCGCTTGGCCTCCGACT TNNNNNNNN-3'

Downstream random primer: 5'-P-NNNNNNNN GTCTCCAGTCGAAGCCCGA CG-ddC-3'

In the upstream random primer, "GACCGCTTGGCCTCCGACT" represents the 5' adapter sequence of the sequencing platform in sequence X. The "GAC" before the 5' adapter is a protective base. "NNNNNNNN" represents the random sequence Y, with a spacer base between sequences X and Y. In the downstream random primer, "GTCTCCAGTCGAAGCCCGA" represents the 3' adapter sequence of the sequencing platform in sequence X. The "CG" after the 3' adapter is a protective base. "NNNNNNNN" represents the random sequence Y, with P indicating phosphorylation and ddC indicating dideoxy modification. Random sequences are randomly synthesized and are not specifically limited here.

The synthesized primers were diluted to 10 μM for later use.

2. Genomic DNA sequence denaturation

This example uses a chemical denaturation method. Specifically, extracted human genomic DNA was diluted to 50 ng/µL. The denaturation reaction system was prepared as follows: 1 µL DNA sample, 0.6 _µL ddH₂O, and 1 µL denaturation buffer, for a total of 2.6 µL. Denaturation was then completed by incubating at room temperature for 3 minutes. In this example, the denaturation buffer consisted of 208 mM KOH and 1.3 mM EDTA.

3. Random Primer Annealing

Add 1 μL of neutralization buffer (208 mM HCl, 312.5 mM Tris-HCl) to the denaturation system and incubate at room temperature for 3 minutes. Add 1 μL of annealing solution and the downstream and upstream random primers at a 1:2 ratio to achieve a total concentration of 5.1 pmol.

The annealing reaction solution was prepared as follows: 10x phi buffer 0.46ul, ddH ₂ O 0.03ul, upstream random primer 10uM 0.34ul, downstream random primer 10uM 0.17ul, a total of 1ul.

The reaction conditions were room temperature for 10 minutes.

4. Sequence extension

Add 15.4 µL of extension reaction solution to the above reaction system. The dNTP concentration in the extension reaction solution is 0.85 nmol. The extension reaction solution is prepared as follows: 1.54 µL of 10x phi buffer, 3.56 µL of purified water, 1 µL of dimethyl sulfoxide, 8 µL of 5M betaine, 0.85 µL of 0.25 mM each dNTP, 0.25 µL of 2 U/µL DNA polymerase, and 0.2 µL of 400 U/µL DNA ligase, for a total of 15.4 µL.

Extension conditions are related to the appropriate library size for the sequencing platform. In this example, extension is performed at 37°C for 20 minutes, followed by a 15-minute reaction at 65°C to heat-inactivate the DNA polymerase. It should be noted that since the size of randomly fragmented fragments is determined by the molar ratio of the sum of the upstream and downstream random primers to the DNA sample, it is understandable that larger fragments require longer extension times, while smaller fragments require shorter extension times. Therefore, extension conditions are related to fragment size, and therefore library size.

5. Purification of Ligation Products

The single-stranded ligation product was purified using magnetic beads. In this example, 1.0x PEG32 magnetic beads were used. 30 μL of PEG32 magnetic beads were added to the 30 μL ligation system described above to purify the single-stranded ligation product. The product was then dissolved in pure water to obtain single-stranded, randomly interrupted DNA.

6. PCR reaction

Amplification was performed using the purified randomly interrupted DNA as a template, and primer sets were designed for the 5' end adapter and 3' end adapter in the upstream random primer and the downstream random primer. The forward primer in the primer set was shown in Seq ID No. 4, and the reverse primer was shown in Seq ID No. 5.

Seq ID No.4: 5'-TCCTAA GACCGCTTGGCCTCCGACT -3'

Seq ID No.5: 5'-AGACAAGCTCGA TCGGGCTTCGACTGGAGAC -3'

It should be noted that, compared with the forward primer of the sequence " GACCGCTTGGCCTCCGACT " shown in Seq ID No.1 and the reverse primer of the sequence " TCGGGCTTCGACTGGAGAC " shown in Seq ID No.3, the 5' ends of the forward primer and reverse primer in this example were respectively added with the adapter of the second sequencing platform, thereby obtaining the forward primer and reverse primer shown in Seq ID No.4 and Seq ID No.5; it can be understood that the adapter of the second sequencing platform added to the 5' end does not affect the amplification, therefore, the forward primer and reverse primer shown in Seq ID No.1 and Seq ID No.3 are also applicable to this example.

PCR reaction system: 20.5ul of purified single-stranded DNA, 25ul of 2X PCR buffer, 2ul of 20uM forward primer, 2ul of 20uM reverse primer, 0.5ul of 400U/ul DNA polymerase, a total of 50ul.

The PCR reaction conditions were denaturation at 95°C for 3 min, followed by 15 cycles of reaction: 95°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. After the cycle was completed, the reaction was extended at 72°C for 10 min, and then incubated at 4°C.

7. Sequencing Verification

The PCR products were sequenced by Illumina Hiseq2000 PE101. After filtering, the reads obtained by sequencing were aligned with the reference genome sequence. The amount of alignment data and the genome coverage at different depths were counted. The results are shown in Table 1.

Table 1 Coverage and uniformity distribution of sequencing data aligned back to the reference genome

Number of sequencing read lengths (M) 4.5 Matching rate 99.70% Number of uniquely aligned reads (M) 4.5 Genome coverage 99.99% Genome coverage at 4X depth 99.89% Genome coverage at 10X depth 98.53% Genome coverage at 20X depth 96.25% Genome coverage at 30X depth 85.20% Genome coverage at 40X depth 73.55% Genome coverage at 50X depth 55.60%

The alignment rate shows that the fragments amplified by the double random primers in this example are basically all of the target species, with good specificity; the genome coverage shows that the target genome is basically fully covered, indicating that the random interruption method in this example has good randomness; the genome coverage at different depths shows that the coverage uniformity in this example is relatively good, and most areas are deeply covered, such as 4X and 10X, which can meet the needs of subsequent variation analysis.

Based on the above experiments, this example further optimized the number of different random sequences and the ratio of the upstream random primer amount to the downstream random primer amount. At the same time, a comparative test was conducted on three polymerases; the results are shown in Figure 2. In Figure 2, lane D2000 is the electrophoresis result of DNA marker, lane 1 is the electrophoresis result of 5 random base sequences, downstream random primer: upstream random primer = 1:1, using Taq DNA polymerase; lane 2 is the electrophoresis result of 5 random base sequences, downstream random primer: upstream random primer = 2:1, using Taq DNA polymerase; lane 3 is the electrophoresis result of 5 random base sequences, downstream random primer: upstream random primer = 3:1, using Taq DNA polymerase; lane 4 is the electrophoresis result of 5 random base sequences, downstream random primer: upstream random primer = 3:1, using E.Coli DNA polymerase I; lane 5 is the electrophoresis result of 5 random base sequences, downstream random primer: upstream random primer = 3:1, using klenow Lane 6 is the electrophoresis result of Klenow Fragment with 9 random base sequences, downstream random primers:upstream random primers=2:1; lane 7 is the electrophoresis result of Klenow Fragment with 8 random base sequences, downstream random primers:upstream random primers=2:1; lane 8 is the electrophoresis result of Klenow Fragment with 5 random base sequences, downstream random primers:upstream random primers=2:1; lane 9 is the electrophoresis result of Klenow Fragment with 6 random base sequences, downstream random primers:upstream random primers=2:1; lane 10 is the electrophoresis result of Klenow Fragment with 7 random base sequences, downstream random primers:upstream random primers=2:1. The results showed that the comparison of the three polymerases showed that the klenow fragment was more effective, and the comparison of the number of random base sequences showed that the random anchor PCR with 8 random bases was more effective. The ratio of downstream random primers to upstream random primers could be between 1 and 3:1, and the ratio of 2:1 was better.

On this basis, this example further tested the total amount of upstream random primers and downstream random primers, and the results are shown in Figure 3. In Figure 3, lane D2000 is a DNA marker; lane 1 is a negative control; lane 2 is the electrophoresis result of Klenow Fragment using a ratio of downstream random primers to upstream random primers of 2:1, with a total amount of random primers of 200 pmol; lane 3 is the electrophoresis result of Klenow Fragment using a ratio of downstream random primers to upstream random primers of 2:1, with a total amount of random primers of 51 pmol; lane 4 is the electrophoresis result of Klenow Fragment using a ratio of downstream random primers to upstream random primers of 2:1, with a total amount of random primers of 5.1 pmol; lane 5 is the electrophoresis result of Klenow Fragment using a ratio of downstream random primers to upstream random primers of 2:1, with a total amount of random primers of 0.51 pmol. The results showed that a total amount of 5.1 pmol of random primers was optimal. This means that, based on a DNA sample of m = 50 ng and fragmentation to L = 300 bp, the theoretical primer dosage n = 0.253 pmol. R = 5.1/0.253 is approximately 20, meaning the actual amount of upstream and downstream random primers required to fragment 50 ng of DNA to 300 bp is 20 times the theoretical amount. Extensive experimental research was conducted to determine the value of R. The results showed that, based on the library construction process, specifically the fragment size required, and depending on the fragment length L, R ranged from 2.7 ≤ R ≤ 750. The smaller L, the higher the multiple of the theoretical amount (i.e., the larger R). An R of 20 is preferred, considering the more conventional fragmentation of DNA samples to 300 bp.

The primers in this example use double random anchoring of upstream random primers and downstream random primers to achieve random fragmentation of the DNA sample. The primer set is then used to amplify the purified randomly interrupted single-stranded DNA fragments to obtain a DNA library that can be applied to different sequencing platforms. The operation is simple and convenient, avoiding dependence on special equipment and expensive kits, and greatly expanding the application field of large-scale high-throughput sequencing.

The above content is a further detailed description of the present application in conjunction with specific implementation methods, and it cannot be considered that the specific implementation of the present application is limited to these descriptions. For ordinary technicians in the technical field to which the present application belongs, they can make several simple deductions or substitutions without departing from the concept of the present application, which should be considered to fall within the scope of protection of the present application.

SEQUENCE LISTING

<110> Shenzhen MGI Technology Co., Ltd.

<120> A primer for random nucleic acid fragmentation and a method for random nucleic acid fragmentation

<130> 17F24115

<160> 5

<170> PatentIn version 3.3

<210> 1

<211> 19

<212> DNA

<213> Artificial sequence

<400> 1

gaccgcttgg cctccgact 19

<210> 2

<211> 19

<212> DNA

<213> Artificial sequence

<400> 2

gtctccagtc gaagcccga 19

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<400> 3

tcgggcttcg actggagac 19

<210> 4

<211> 25

<212> DNA

<213> Artificial sequence

<400> 4

tcctaagacc gcttggcctc cgact 25

<210> 5

<211> 31

<212> DNA

<213> Artificial sequence

<400> 5

agacaagctc gatcgggctt cgactggaga c 31

Claims (6)

1. A method for random fragmentation of nucleic acids, comprising using primers to perform double random anchoring on a DNA sample, wherein the primers consist of a plurality of upstream random primers and a plurality of downstream random primers; specifically comprising: hybridizing the upstream random primers and the downstream random primers to a denatured DNA sample; between the most adjacent upstream and downstream random primers, by the action of DNA polymerase, extending the upstream random primers to the 3' end to fill the sequence between the upstream and downstream random primers; and then, by the action of DNA ligase, ligating the 3' end of the extended sequence of the upstream random primers to the 5' end of the downstream random primers, i.e., ligating the upstream random primers and their extended sequences to the downstream random primers into a single segment, thereby achieving double random fragmentation of the DNA sample through random hybridization of the upstream and downstream random primers; During the process of hybridizing the upstream random primer and the downstream random primer to the denatured DNA sample, the total amount of the upstream random primer and the downstream random primer used is R×n picomoles, where 2.7≤R≤750, n=1.515×(m÷L), m is the weight of the DNA sample in nanograms, L is the expected length of the fragmented DNA, and n is the theoretical amount of upstream and downstream random primers required to break the DNA sample into fragments of length L, in picomoles. The molar ratio of the upstream random primer to the downstream random primer is 1 to 3:1. The sequence composition of the upstream random primer is: 5’-X-Y-3’; The sequence composition of the downstream random primer is: 5’-P-Y’-X’-close-3’; Where Y and Y’ are random sequences, X is all or part of the 5’ adapter sequence of the sequencing platform, X’ is all or part of the 3’ adapter sequence of the sequencing platform, P is phosphorylation modification, and close is a blocking modification used to prevent the formation of 3-5 phosphodiester bonds. In use, the upstream random primer and the downstream random primer hybridize and bind on the same template strand. The upstream random primer extends to the 3' end, filling the gap between the nearest downstream random primer and the upstream random primer. The 3' end of the extended sequence of the upstream random primer is connected to the 5' end of the downstream random primer, thus linking the upstream random primer and its extended sequence with the downstream random primer into a single segment. The blocking modification is a dideoxy modification; The X sequence of the upstream random primer also includes 2-6 protective bases at its 5' end; the X' sequence of the downstream random primer also includes 2-6 protective bases at its 3' end, and the blocking modification is applied above one of the terminal protective bases. The X sequence of the upstream random primer has the sequence shown in Seq ID No. 1, and the X’ sequence of the downstream primer has the sequence shown in Seq ID No. 2; Seq ID No.1: 5’-GACCGCTTGGCCTCCGACT-3’; Seq ID No.2: 5’-GTCTCCAGTCGAAGCCCGA-3’; The 5' end of the X sequence of the upstream random primer is protected with "GAC", and the 3' end of the X' sequence of the downstream random primer is protected with "CG". 2. The method according to claim 1, wherein the molar ratio of the upstream random primer to the downstream random primer is 2:1. 3. The method according to claim 1, wherein R = 20. 4. A method for constructing a nucleic acid library, comprising randomly fragmenting a DNA sample using the method described in any one of claims 1-3, and then performing PCR amplification on the double-randomly fragmented DNA fragments using a pair of universal primers to obtain a nucleic acid library enriched with random fragments; wherein the universal primers consist of a forward primer and a reverse primer, wherein the 3' end of the forward primer has all or part of the sequence of the 5' adapter of the sequencing platform, and the 3' end of the reverse primer has all or part of the reverse complementary sequence of the 3' adapter of the sequencing platform. 5. The construction method according to claim 4, characterized in that: the 5' ends of the forward primer and the reverse primer respectively have adapter sequences of the second sequencing platform. 6. The construction method according to claim 4, wherein the forward primer contains the sequence shown in Seq ID No. 1, and the reverse primer contains the sequence shown in Seq ID No. 3; Seq ID No. 3: 5’-TCGGGCTTCGACTGGAGAC-3’.