CN111575272B

CN111575272B - High-copy DNA repetitive sequence in vitro rapid synthesis based on blocking type chain polymerization amplification reaction

Info

Publication number: CN111575272B
Application number: CN201911265744.5A
Authority: CN
Inventors: 杨雪瑞; 王鑫
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2021-09-17
Anticipated expiration: 2039-12-11
Also published as: CN111575272A

Abstract

The present invention provides a method for synthesizing a DNA sequence comprising a repeating unit, the method comprising designing and synthesizing an extension primer and a blocking primer based on the repeating unit, and obtaining the DNA sequence comprising a repeating unit by performing a PCR amplification reaction using the repeating unit (as an amplification template), the extension primer and the blocking primer in a PCR reaction system. The invention also provides a kit for use in the method. The method has the characteristics of controllable copy number of the synthetic repetitive sequence, simple synthetic steps, low cost and the like, and is very suitable for industrial high-throughput production.

Description

High-copy DNA repetitive sequence in vitro rapid synthesis based on blocking type chain polymerization amplification reaction

Technical Field

The invention relates to the technical field of DNA amplification, in particular to a method for synthesizing a DNA sequence comprising a repeating unit. The invention also provides a kit for use in the method.

Background

In recent years, with the improvement of sequencing capability, genome information of a plurality of species is cracked, a large number of repeated DNA sequences are discovered in succession, the content of the repeated DNA sequences is positively correlated with the species evolution, and the types and the contents of the repeated DNA sequences in the genome of a eukaryote are far greater than those of prokaryotes, so that the method indicates that repeated DNA elements are directly correlated with the biological evolution and the complex biological function, and has very strong biological function and research significance. With the deepening of genomics research, people find that DNA double-stranded sequences with different copy numbers have obvious difference on the recombination kinetic characteristics, the content of the repetitive units and the copy number have a negative correlation with the recombination time, and therefore the DNA repetitive sequences are divided into single-copy repetitive sequences according to the kinetic characteristics; a low degree of repeat sequence; medium repetitive sequences and high repetitive sequences. The medium and high repetitive sequence elements are directly related to biological functions such as chromosome construction, structure maintenance and gene expression regulation. In addition, because the medium-high-degree repeated DNA molecules have strong affinity self-assembly capability, the medium-high-degree repeated double-stranded DNA molecules are often also excellent basic building molecules of biocompatible materials.

In summary, highly repetitive DNA sequences in vitro artificial synthesis have very important physiological and material science application values, and the existing methods for in vitro synthesis of moderately and highly repetitive DNA are limited and mainly use RCA (rolling circle amplification) and gene synthesis splicing techniques.

The RCA technology mainly uses the cyclization amplification principle of viruses and prokaryotes plasmids for reference, a specific single-stranded DNA molecule is subjected to cyclization connection through ligase to obtain a cyclization DNA template molecule, and then a single-stranded DNA primer matched with the cyclization template and a high-efficiency single-stranded amplification enzyme (phi29) are used for circularly replicating the cyclization template under an isothermal environment (25-37 ℃), so that a repeated single-stranded DNA sequence which is complementarily matched with the cyclization template is obtained (shown in figure 1). The method requires cyclization through a single-stranded template; the method has the advantages that the steps of non-circular single-stranded DNA elimination and circular amplification are three, the operation steps are complex, the cost of bioactive enzymes required by each technical circle is high, the industrialization of the method is severely limited, in addition, the final product is a single-stranded repeated DNA molecule, and more single-stranded primers need to be additionally added in the later-stage improved method MCH, so that a DNA double-stranded molecule product is obtained. The RCA method obtains the positive correlation between the copy number of the repeated DNA molecules and the reaction time, namely the copy number of the final product can be controlled only by controlling the reaction time, the product length is difficult to predict in the actual operation, the copy number of the product repeated sequence is difficult to accurately control, and the practicability is lacked, so that the high-degree repeated DNA in the synthesis of the RCA amplification method is always stopped at the application stage in a laboratory, and the industrialization is difficult.

With the maturation of high-throughput DNA primer synthesis technology, overlap region amplification Gene splicing is becoming the mainstream of Gene synthesis (Gene SOE). The Gene SOE technology divides a long DNA fragment into multiple levels of small fragments, specific recognition base sequences exist on two sides of each fragment, sequential assembly of the multiple levels of small fragments is completed under the cooperation of ligase or amplificase by the base complementary principle, and finally a long fragment DNA Gene sequence is obtained (as shown in figure 2). The length of the base in the overlapping region, the base ratio and the Tm value of the primer have important influence on the whole splicing efficiency. However, when the copy number of the highly repetitive DNA sequence reaches a certain threshold, base pairing slippage and mismatching can occur inside the DNA molecule, which seriously affects the amplification and ligation efficiency, resulting in problems of decreased splicing efficiency, increased error rate, and the like, and finally resulting in problems of long period of highly repetitive DNA in the synthesis by the splicing method, high base mutation rate, difficult control of copy number, and the like, and finally affecting the synthesis of the repetitive DNA double strand.

Disclosure of Invention

In order to solve the problems of high cost, long period, high error rate and the like in the synthesis of the repetitive sequence, the inventor develops a novel method for quickly synthesizing the high-degree repetitive sequence in vitro.

The method of the present invention is a block PCR based Repeat amplification technology (BPRE), which is based on the common chain polymerase amplification reaction, using three complementary paired DNA primer sequences, Blocking the primer template and the Repeat unit template to realize steric hindrance pairing in the cyclic heating denaturation process, exposing several (for example, 6) bases at the 3 'end of the Repeat unit primer, pairing the exposed several free base groups and the complementary bases at the 3' end of the amplification primer (extension primer) template to form primer dimer formation, completing the bidirectional amplification of the dimer under the action of the amplification enzyme (Taq DNA polymerase), and completing the accumulation extension of a Repeat sequence (as shown in FIG. 3). The newly generated repeated DNA double-chain sequence is subjected to the heating denaturation and cooling renaturation processes again, the steric hindrance and dimer generation extension processes are repeated, and the accumulative increase of the highly repeated DNA sequence is finally completed, so that the accumulative amount of the repeated sequence of the method is in positive correlation with the number of heating and cooling cycles.

Accordingly, the present invention provides the following:

1. a method of synthesizing a DNA sequence comprising a repeat unit, the method comprising the steps of:

1) designing and synthesizing an extension primer and a blocking primer based on the repeat unit, wherein the blocking primer differs from the complement of the repeat unit by n nucleotides of the 5 'end (5' to 3 'direction) lacking the complement sequence, the extension primer differs from the complement of the repeat unit by the addition of the n nucleotides of the 3' end (5 'to 3' direction) such that both the extension primer and the blocking primer are exactly the complement sequences of two of the repeat units when connected in series in the 5 'to 3' direction, wherein n is an integer from 3 to 20, preferably from 4 to 10 (e.g., 4, 5, 6, 7, 8, 9, and 10); and

2) in the PCR reaction system, the DNA sequence comprising a plurality of repeating units is obtained by performing a PCR amplification reaction using the repeating unit (as an amplification template), an extension primer (as a repeating unit extension template), and a blocking primer. In the PCR reaction system, a blocking primer is first paired with a repeating unit (since the blocking primer differs from the complementary sequence of the repeating unit in that n nucleotides of the 5 ' end of the complementary sequence are absent (in the 5 ' to 3 ' direction), the n nucleotides of the 3 ' end of the repeating unit are in an unpaired state), and then the n nucleotides of the 3 ' end of an extension primer are paired with unpaired nucleotides on the repeating unit, followed by PCR extension amplification reaction.

The "DNA sequence comprising a plurality of repeating units" according to the present invention is also referred to as a repeat sequence. The repetitive sequences are widely present in the genome and can be divided into two major categories, namely tandem repetitive sequences and interspersed repetitive sequences, according to the arrangement mode of the repetitive units. The tandem repeat sequence is a repeat sequence in which repeat units are connected end to end and concatenated together. The method of the invention is particularly suitable for the synthesis of tandem repeat sequences. The length of the Repeating Unit (RU) is not particularly limited, and may be, for example, 3 to 100 (e.g., 20, 30, 40, 50, 60, 70, 80, 90 or 100) nucleotides, preferably 10 to 30 nucleotides, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides.

2. The method according to 1 above, further comprising the step of determining the copy number of said repeat unit in the obtained DNA sequence, preferably by analyzing the molecular weight of the PCR product by gel electrophoresis or by DNA sequencing techniques. The copy number of the repeating unit in the DNA sequence obtained in the present invention may be 2 copies to several thousands copies, for example, 5 to 200 copies, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 copies.

3. The method according to 1 above, wherein a PEG molecule is further included in the PCR reaction system, preferably having a molecular weight of 2000-20,000 Da, more preferably 4,000Da, 6,000Da or 8,000Da, and more preferably having a concentration of 4 wt% to 20 wt% (e.g., 4 wt%, 8 wt% and 12 wt%) in the PCR reaction system.

4. The method according to the above 3, wherein NaCl is further included in the PCR reaction system. In a preferred embodiment of the invention, the effect of adding different concentrations of NaCl on the reaction product is tested when using a new batch of primers, and the optimal concentration is selected. Preferably, the concentration of NaCl used in the PCR reaction system is 20 to 80mM, preferably 30 to 60 mM.

5. The method according to 1 above, wherein the molar ratio of the repeating unit, the extended primer and the blocked primer in the PCR reaction system is 1:1 to 10:1 to 40, preferably 1:1: 10.

6. The method of 1 above, wherein the PCR amplification reaction is a two-step PCR or a three-step PCR.

The PCR reaction system of the present invention may perform the PCR reaction by any suitable PCR method known in the art.

Standard three-step PCR typically comprises the following steps:

1) DNA denaturation: (90 ℃ -96 ℃): the double-stranded DNA template is broken by hydrogen bonds under the action of heat to form single-stranded DNA;

2) annealing: (60 ℃ -65 ℃): the temperature of the system is reduced, and the primer is combined with the DNA template to form a local double strand;

3) extension: (70 ℃ to 75 ℃): under the action of Taq enzyme (about 72 ℃ C., the activity is optimal), dNTP is used as a raw material, and a DNA strand complementary to the template is synthesized by extending from the 3 ' -end of the primer in the direction from the 5 ' → 3 ' -end.

For the copy number of the PCR product, fluorescein (ethidium bromide, EB) stained gel electrophoresis is the most commonly used detection means. Electrophoresis detection methods are also useful detection methods. In addition, fluorescent probe detection methods are available.

The present invention preferably employs two-step PCR, which differs from standard three-step PCR mainly in that:

1) the method comprises the following steps: annealing and extension are simultaneously carried out by a two-step method so as to reduce the temperature rise and drop process, and the three-step method is completed by two times;

2) the use time is different: the two-step method reduces the one-time temperature rise and fall process, improves the reaction speed and has shorter time consumption.

7. The method according to any one of 1 to 6 above, wherein the DNA sequence comprising the repeating unit is a low repeating sequence, a medium repeating sequence or a high repeating sequence, preferably a medium repeating sequence or a high repeating sequence.

8. A kit for synthesizing a DNA sequence comprising a repeat unit, the kit comprising a PCR reaction system comprising the repeat unit for use as an amplification template, an extension primer and a blocking primer, wherein the blocking primer differs from the complement of the repeat unit by the absence of n nucleotides from the 5 'end of the complement sequence (in the 5' to 3 'direction), the extension primer differs from the complement of the repeat unit by the addition of the n nucleotides to the 3' end (in the 5 'to 3' direction), such that both the extension primer and the blocking primer are exactly the complement of two of the repeat units when connected in series in the 5 'to 3' direction, wherein n is an integer from 3 to 20, preferably from 4 to 10 (e.g., 4, 5, 6, 7, 8, 9 and 10).

9. The kit according to 8 above, wherein:

1) also included in the PCR reaction is a PEG molecule, preferably having a molecular weight of 2000-20,000 Da, more preferably 4,000Da, 6,000Da or 8,000Da, more preferably having a concentration of 4 wt% to 20 wt% (e.g., 4 wt%, 8 wt% and 12 wt%) in the PCR reaction;

2) NaCl is also included in the PCR reaction system. When a new batch of primers is used, the influence of adding NaCl with different concentrations on the reaction product is tested, and the optimal concentration is selected. Preferably, the using concentration of NaCl in the PCR reaction system is 20-80 mM, preferably 30-60 mM;

3) the molar ratio of the repeating unit to the extension primer to the blocking primer in the PCR reaction system is 1:1-10:1-40, preferably 1:1: 10; and/or

4) The PCR reaction system comprises Taq DNA polymerase.

10. The kit according to 8 or 9 above, wherein the DNA sequence comprising a repeating unit is a low repeating sequence, a medium repeating sequence or a high repeating sequence, preferably a medium repeating sequence or a high repeating sequence.

The method can indirectly control the copy number of the repeated sequences by controlling the number of heating and cooling cycles, has the characteristics of controllable copy number of the repeated sequence synthesis, simple synthesis steps and the like, and is very suitable for industrial high-throughput production. In addition, the synthesis method only needs a single biological enzyme active substance, the three initial single-stranded DNA primers are few in demand, a reaction system is trace, the purity of the primers is not required (NaCl concentration can be used for adjusting and calibrating the reaction efficiency in the synthesis process), the preparation cost is greatly reduced due to the fact that the existing repeated sequence synthesis method is far from the economic benefit, and the method has obvious cost advantage compared with the existing method.

Drawings

The above features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a schematic representation of a prior art RCA (Rolling circle amplification) technique;

FIG. 2 is a schematic diagram of the prior art Gene SOE technique;

FIG. 3 is a schematic diagram of the Blocking PCR based repetitive amplification technique (Blocking PCR based Repeat Expansion (BPRE)) of the present invention;

FIG. 4 is a specific embodiment of a BPRE according to the present invention;

FIG. 5 shows the effect of the molar ratio of RU, TP and BP on the reaction in the PCR reaction system in the method of the present invention (the ratio in the figure is the molar ratio of RU: TP: BP);

FIG. 6 shows the effect of PEG molecular weight (A) and PEG concentration (B) on the reaction in the PCR reaction system in the method of the present invention;

FIG. 7 shows the effect of NaCl solution concentration on the reaction in the PCR reaction system in the method of the present invention;

FIG. 8 shows the relationship between the number of cycles of warming and cooling of a double-stranded repetitive DNA product in the method of the present invention (numerals 20, 40, 70, 90 and 120 in the figure indicate the number of cycles of warming and cooling in a PCR reaction);

FIG. 9 shows determination of the copy number of the repeating unit included in the obtained PCR product (double-stranded repetitive DNA product) by the gel electrophoresis method and the sequencing method; and

FIG. 10 shows that the PCR product (double-stranded repetitive DNA product) obtained by the method of the present invention can be used as a template, and after further PCR amplification and treatment, a DNA affinity gel (A) is obtained, which can be used as a drug release material (B, release profile of doxorubicin).

Detailed Description

Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art.

The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The chemicals used in the following reactions are all commercially available products unless otherwise indicated.

Example 1 design of primers

TP (extension primer), BP (blocking primer), and related sequences (beka bosch biotechnology limited) were designed according to the base sequence composition of RU (repeat unit) (as shown in fig. 4), respectively, and synthesized as single-stranded DNA primers, with the maximum raw material primer requirement of 1OD (about 33 micrograms dry powder) and the minimum raw material primer requirement of 0.1OD (about 3.3 micrograms dry powder).

RU:5’-GGAGAGCTGTGGACTTCGTC-3’(SEQ ID NO:1)

TP:5’-GACGAAGTCCACAGCTCTCCGACGAA-3' (base pairing of TP and RU is underlined) (SEQ ID NO:2)

BP:5’-GTCCACAGCTCTCC-3’(SEQ ID NO:3)

Example 2 influence of molar ratios of RU, TP and BP on the reaction in a PCR reaction System

Dissolving the primer dry powder to 100 mu M by using distilled water, and mixing RU: TP: BP three primers to form a mixed stock solution, wherein the final concentration of RU in the mixed solution is 4 mu M. The PCR instrument is an Eppendorf PCR instrument. The PCR reaction used Taq DNA polymerase (Taq, Vazyme, P515-02). The three-primer stock solution and an amplification enzyme solution, a PEG4K (solarbio, P8240) solution and a NaCl solution are diluted and mixed until the final concentration of the RU primer is 400nM, the amplification enzyme is 1x working concentration, and the final reaction system range is 10-20 muL.

After the amplification reaction system is configured, performing cycle nested temperature rise and fall amplification operation to synthesize repeated double-stranded DNA molecular fragments, wherein the PCR cycle heating condition is as follows:

95 ℃ for 2 minutes

95 ℃ for 15 seconds

70 ℃ for 15 seconds

5 minutes at 72 DEG C

Because the directional amplification is carried out by adopting the short-time heating and cooling amplification method, the reaction preparation time is generally within 1-2 hours.

Different RUs: TP: the molar ratio of BP had a significant effect on amplification efficiency and product bands, as shown in fig. 5, RU: TP: the optimal molar ratio of BP is 1:1:10, the product band is obvious (DNA marker; DSBIO; Low Ladder) at the ratio, and the specificity is higher. Thus, the following experiments all used a molar ratio of 1:1:10 of RU: TP: BP.

Example 3 Effect of molecular weight and concentration of PEG in PCR reaction System on the reaction

In the PCR reaction system, PEG can be used as a synergist of PCR reaction, so that the amplification efficiency is improved.

A in FIG. 6 shows the catalytic effect of the PCR reaction of solutions of PEG with different molecular weights (solarbio) at the same concentration (4 wt%), as shown in the figure, PEG molecules with 4-8K molecular weight can catalyze complementary base pairing (DNAmarker; Tiangen; D2000), thereby promoting the amplification extension reaction, and the amplification product can hardly be obtained by PEG molecules with molecular weight exceeding 20K.

On the other hand, B in FIG. 6 shows that when the concentration of PEG (PEG4K) in the reaction system is less than 4 wt% (DNAmarker; DSBIO; Low Ladder), the entire extension reaction hardly proceeds, and the entire reaction continues only after the PEG concentration content reaches a certain threshold.

EXAMPLE 4 Effect of NaCl solution concentration on the reaction in PCR reaction System

Because the salt-separation sensitive molecule PEG4K exists in the reaction system, different salt-separation concentrations can regulate the amplification efficiency of DNA products. The residual quantity of the salt solution in the DNA primers synthesized in different batches has batch difference, so in order to ensure the consistency of amplification efficiency among the batches and obtain the optimal amplification effect, the salt concentration sensitivity test can be carried out on the single-stranded primers in different batches, and the optimal salt ion concentration of the batch is selected according to the experimental result. We used NaCl solution as calibration reagent, using the same concentration of 4 wt% PEG4K, using different concentrations of NaCl for the previous experiments. As shown in FIG. 7, in this experimental system, the amplification band was single under the condition of adding 60mM NaCl solution compared with other conditions, and the PCR amplification reaction was inhibited above this concentration; at concentrations below this, smearing of the amplification product is more pronounced and the product size is severely distorted (DNAmarker; DSBIO; Lowladder).

Example 5 relationship between the length of double-stranded DNA repeat and the number of cycles of warming and Cooling

This example shows the relationship between the number of copies of the repeat unit contained in the obtained PCR product and the number of cycles of warming and cooling of the PCR reaction.

The results are shown in FIG. 8, demonstrating that the number of cycles is positively correlated with the number of copies of the repeat unit (repeat sequence). When the number of amplification cycles is low, only 2-3 repetitive DNA double-stranded molecules are amplified. When the number of amplification cycles increases to 90 cycles, 20 copies of the repetitive double-stranded DNA molecule are generated, and the high-copy repetitive sequence DNA product is relatively single (DNAmarker; DSBIO; Low Ladder).

The prepared double-stranded repetitive DNA product can be connected to a common carrier plasmid for sequencing detection and permanent storage, and the amplification product can also be directly used for other experiments.

Example 6 determination of the number of copies of the repeat Unit in the repetitive double stranded DNA

The repetitive DNA sequence obtained in the above example was ligated to pUC19 blue white spot screening system (pUC 19 plasmid provided in Tiangen DH 5. alpha. competent cells), and subjected to sequencing test (Beijing Rui Boxing Ke Biotechnology Co., Ltd.) to obtain a DNA double-stranded molecule of 45 copies of repetitive units without any base mutation, the whole preparation process took about 1 hour, and the molecular weight of the prepared DNA was as indicated by gel electrophoresis. The cost of the consumable materials for preparing the reagents is about RMB 30 yuan.

The synthesized DNA double strand with 45 copy repeat units has a CAG.CTG repeat level structure inside, a single strand internal sliding structure is very easy to form, the synthesis difficulty is greatly increased, and the synthesis period or the DNA base error rate can be greatly prolonged by using the existing DNA synthesis method (such as the Gene SOE technology). As shown in FIG. 9, by electrophoresis and sequencing verification (Beijing Rui Boxing Ke Biotechnology Co., Ltd.), the method of the present invention can maximally avoid the problem by synthesizing DNA repeat sequences in a blocking manner while synthesizing, thereby obtaining medium and high copy DNA repeat sequences without any base mutation and error.

Example 7 repeated double stranded DNA formation of DNA affinity gel and use in drug sustained release

The double-stranded DNA prepared in example 6 was used as a template, RU and TP were used as primers for ordinary PCR amplification, and the amplified product was purified, heat-shocked and cooled at 85 ℃ and then cross-linked between DNA molecules to form a DNA affinity gel (as shown in FIG. 10, A, the lower left is an image of the DNA gel under the DNA dye gelspace (Yuanhinghao Bio)). The generated DNA gel has good ductility and has good slow release function (such as adriamycin) on DNA affinity molecules. FIG. 10, B is a 7-day sustained release profile of doxorubicin (Yuanye Bio-Technology; S17092-25mg) according to the experimental procedure in which a doxorubicin solution (0.125. mu.g) was added during the preparation of the DNA gel, the prepared gel was incubated in ultra-pure water at 37 ℃ and the doxorubicin-specific absorption peak at 490nm in the solution was detected every 24 hours using a microplate reader (SpectraMax M5). The experiment result shows that the DNA gel can maintain the drug slow release effect for 5 days and has good drug slow release function.

In conclusion, the blocking type PCR method for synthesizing the high-degree repetitive sequence is a synthetic method for blocking the synthesized repetitive sequence and adding new copies, has the characteristics of low synthetic cost, simple operation, short time consumption, no need of complex instruments and the like, the whole reaction system is completely based on the traditional DNA chain type amplification system, only a single bioactive molecule is needed in the reaction process, the cost is further reduced, the whole process is carried out in a distribution sectional mode, the product length and the copy number can be controlled through the number of reaction cycles, and the method has obvious advantages compared with the existing synthetic method.

Industrial applicability

The copy number of the repeated DNA molecules is directly related to a plurality of biological functions and molecular materials, such as chromosome structure regulation, cell canceration, species evolution, DNA affinity gel, DNA self-contained nano-structures and the like. The simple and efficient preparation of the repeat sequence of any DNA sequence and the random copy of the repeat sequence have important biological and material values and wide market demands.

It will be appreciated by persons skilled in the art that although the invention has been described with reference to specific embodiments thereof, the invention is not limited to these specific embodiments. Based on the teaching of the present invention and the technical solutions, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and thus the resulting equivalent embodiments are within the scope of the present invention.

Claims

1) designing and synthesizing an extension primer and a blocking primer based on the repeat unit, wherein the blocking primer differs from the complement of the repeat unit by n nucleotides from the 5 'end of the missing complement, the extension primer differs from the complement of the repeat unit by the addition of the n nucleotides from the 3' end, such that both the extension primer and the blocking primer when concatenated in the 5 'to 3' direction are exactly the complement of both the repeat units, wherein n is an integer from 3 to 20; and

2) obtaining the DNA sequence including the plurality of repeating units by performing a PCR amplification reaction using the repeating units, the extension primers, and the blocking primers in a PCR reaction system,

wherein the length of the repeating unit is 10-100 nucleotides;

wherein the PCR reaction system also comprises PEG molecules, the molecular weight of the PEG molecules is 2000-20,000 Da, and the concentration of the PEG molecules in the PCR reaction system is 4 wt% to 20 wt%;

wherein the PCR reaction system also comprises NaCl, and the use concentration of the NaCl in the PCR reaction system is 20-80 mM; and is

Wherein the molar ratio of the repeating unit to the extension primer to the blocking primer in the PCR reaction system is 1:1-10: 1-40.

2. The method of claim 1, wherein n is an integer from 4 to 10.

3. The method of claim 2, wherein n is 4, 5, 6, 7, 8, 9, or 10.

4. The method of claim 1, further comprising the step of determining the copy number of the repeat unit in the obtained DNA sequence.

5. The method of claim 4, wherein the molecular weight of the PCR product is analyzed by gel electrophoresis or the copy number of the repeat unit in the obtained DNA sequence is determined by DNA sequencing techniques.

6. The method of claim 1, wherein the PEG molecule has a molecular weight of 4,000Da, 6,000Da, or 8,000 Da.

7. The method of claim 1, wherein the concentration of the PEG molecule in the PCR reaction system is 4 wt%, 8 wt%, or 12 wt%.

8. The method according to claim 1, wherein NaCl is used in the PCR reaction system at a concentration of 30 to 60 mM.

9. The method of claim 1, wherein the molar ratio of the repeat unit, the extension primer and the blocking primer in the PCR reaction system is 1:1: 10.

10. The method of claim 1, wherein the PCR amplification reaction is a two-step PCR or a three-step PCR.

11. The method of any one of claims 1 to 10, wherein the DNA sequence comprising repeat units is a low repeat sequence, a medium repeat sequence, or a high repeat sequence.

12. The method of claim 11, wherein the DNA sequence comprising repeat units is a medium repeat sequence or a high repeat sequence.

13. A kit for synthesizing a DNA sequence comprising a repeat unit, the kit comprising a PCR reaction system comprising the repeat unit for use as an amplification template, an extension primer and a blocking primer, wherein the blocking primer differs from the complement of the repeat unit by n nucleotides from the 5 'end of the missing complement, the extension primer differs from the complement of the repeat unit by the addition of the n nucleotides from the 3' end such that both the extension primer and the blocking primer when concatenated in the 5 'to 3' direction are exactly the complement of both the repeat units, wherein n is an integer from 3 to 20,

wherein the length of the repeating unit is 10-100 nucleotides;

14. The kit of claim 13, wherein n is an integer from 4 to 10.

15. The kit of claim 14, wherein n is 4, 5, 6, 7, 8, 9, or 10.

16. The kit of claim 13, wherein the PEG molecule has a molecular weight of 4,000Da, 6,000Da, or 8,000 Da.

17. The kit of claim 13, wherein the concentration of the PEG molecule in the PCR reaction system is 4 wt%, 8 wt%, or 12 wt%.

18. The kit according to claim 13, wherein NaCl is used in a concentration of 30 to 60mM in the PCR reaction system.

19. The kit according to claim 13, wherein the molar ratio of the repeating unit, the extension primer and the blocking primer in the PCR reaction system is 1:1: 10.

20. The kit of claim 13, wherein:

the PCR reaction system comprises Taq DNA polymerase.

21. The kit of claim 13, wherein the DNA sequence comprising repeat units is a low repeat sequence, a medium repeat sequence, or a high repeat sequence.

22. The kit of claim 21, wherein the DNA sequence comprising a repeat unit is a medium repeat sequence or a high repeat sequence.