A method for generating short tandem repeats on the coding gene of a recombinant plasmid through in vitro amplification

Abstract

The present invention discloses a method for generating short tandem repeat sequences on the coding gene of a recombinant plasmid through in vitro amplification. The method of the present invention first uses a single-stranded starting primer with a half-repeat unit to guide the starting PCR amplification of the recombinant plasmid, and then uses a pair of double-copy complementary primer pairs to guide the subsequent PCR amplification of the recombinant plasmid. The method of the present invention has successfully generated short tandem repeat sequences on the GPLD1 and INPP4B genes of the recombinant plasmid. The method of the present invention is easy to implement, does not require complex cloning operations, and has a high positive clone ratio. The generated tandem repeat sequences have the following characteristics: (1) The repeat units have the same direction and variable copy numbers. (2) There are no base mutations between the repeat unit connection regions, and the coding gene reading frame is not changed. The method of the present invention is suitable for generating short tandem repeat sequences on any coding gene of the recombinant plasmid, and provides a new research tool for clarifying the relationship between tandem repeat sequences and the pathogenesis of cell senescence, tumorigenesis, and various neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to a method for in vitro generating short tandem repeat sequences, which first uses a single-stranded starting primer with a half-repeat unit to initiate PCR amplification of a recombinant plasmid, and then uses a pair of double-copy complementary primer pairs to guide subsequent PCR amplification of the recombinant plasmid, with a 20-100 bp fragment encoding a gene on the recombinant plasmid as the repeat unit, belonging to the field of biotechnology. Background Art

[0002] In the human genome, repetitive sequences account for more than 50% and are dispersed throughout the genome. Repetitive sequences can be divided into two major categories: tandem repetitive sequences and dispersed repetitive sequences. According to the length of the repeat unit of tandem repetitive sequences, they can be further divided into: microsatellite DNA and minisatellite DNA. The repeat unit length of minisatellite DNA is dozens to hundreds of bp, and the number of repeats is usually dozens to hundreds. Therefore, it is also called variable number of tandem repeats of short fragments. Minisatellite DNA shows high polymorphism in the population due to different numbers of repeats, and it plays an increasingly important role in the fields of life science and medicine.

[0003] Short tandem repeat sequences play an important role in maintaining the stability of the human genome and the occurrence of some genetic diseases. The human chromosomal telomere DNA sequence consists of highly repeated 5'-TTAGGG-3', which is of great significance for protecting chromosomes and controlling cell growth. In tumor cells, the telomere length is significantly shortened and the telomerase activity is significantly increased, indicating that the telomere length is correlated with the occurrence of tumors. Trinucleotide repeats are common repetitive sequences in organisms, and changes in their copy numbers are related to various neuromuscular and neurodegenerative diseases. For example, in patients with Huntington's disease, an increase in the copy number of the CAG trinucleotide repeat sequence of the pathogenic gene IT15 will lead to an earlier onset age and a more severe disease condition. Other common diseases include myotonic dystrophy and fragile X chromosome syndrome, and their pathogenesis is related to an increase in the copy number of trinucleotide repeat sequences. It is speculated that repetitive sequences in the coding region will cause the encoded protein to exhibit cytotoxicity or immunogenicity, and repetitive sequences in the non-coding region will lead to abnormal transcription and translation in the coding region. Therefore, establishing a repeat sequence creation model in vitro and clarifying the mechanism of DNA repeat sequence generation are important ways to reveal the pathogenesis of the above neurodegenerative diseases and provide a theoretical basis for the development of related therapeutic drugs.

[0004] At present, it is considered that the generation mechanisms of tandem repeats in the body mainly include three generation models: slippage replication, rolling circle replication, and telomere replication. (1) The slippage replication mechanism is the generation mechanism of short tandem repeats that has been studied more at present. During DNA replication, the replication of the lagging strand in slippage replication will first form Okazaki fragments, and then the nicks will be ligated. It undergoes disassembly and reassembly of DNA polymerase. During this period, the newly synthesized DNA strand and the template strand will detach from the DNA polymerase. At this time, a loop will form in the template strand or the lagging strand, which will lead to a decrease or increase in the copy number of short tandem repeats. However, this replication model cannot explain the phenomenon that short tandem repeats appear continuously and are amplified in large numbers in the genome, nor the gene mutations existing between the connection regions of short tandem repeats. (2) Rolling circle replication is the replication mode of phage DNA and some plasmid DNA, which can amplify DNA in large quantities in a short time for organisms to use. However, this mechanism is based on the overall replication of phage and plasmid DNA and cannot perfectly explain the process of local small fragment DNA replication in the genome. (3) The replication mechanism of chromosome telomere crawling is the tandem repeat generation mechanism that has been relatively well studied at present. However, telomere replication is a unique replication mechanism that requires telomerase activity and short RNA as the replication template.

[0005] In view of the important role of repetitive sequences in maintaining genome stability and neurodegenerative diseases, people have been developing methods for generating tandem repeats in vitro, especially for generating short tandem repeats in vitro. Currently, the relatively mature methods are template-free PCR amplification and long iterative nucleotide synthesis (abbreviated as SLIP). (1) The template-free PCR method uses a pair of complementary fragments as repeat units. Since the complementary fragments can form misaligned complements, it is possible to extend the length of the repeat fragment after each PCR cycle. This method requires complex cloning and subcloning gene operations in the follow-up to obtain the required short fragment repeat sequence. (2) Although SLIP synthesizes short tandem repeats on plasmids, it requires different restriction enzyme sites at both ends of the repeat sequence. The recombinant plasmid is digested by two different restriction enzymes respectively, and the digested products are mixed and annealed, which may form a plasmid with misaligned complements. Then, DNA polymerase is used to fill the gap region, resulting in an increase in the length of the repeat sequence. After that, it is transformed into competent Escherichia coli, and the transformed plasmid is extracted to obtain a plasmid with an extended repeat fragment. To increase the length of the repeat unit, the above steps need to be repeated for each repeat unit extension cycle. Therefore, although this method does not require gene cloning operations, the generation method is very cumbersome, time-consuming and laborious.

[0006] In view of the significant deficiencies in the above-mentioned methods for in vitro generation of short tandem repeats, the present invention has established a dual-copy complementary primer pair-guided method, which prepares short tandem repeats by in vitro amplification of recombinant plasmids. The key of this method lies in first using a single-stranded starting primer of a semi-repeat unit to guide the initial PCR amplification of the recombinant plasmid, and then using a pair of dual-copy complementary primer pairs to guide the subsequent PCR amplification of the recombinant plasmid. This method is easy to implement and does not require complex cloning and subcloning operations. The positive rate of plasmid clones generating short fragments is high (about 40%). The generated repeat sequences have the following characteristics: (1) The repeat units have the same direction and variable copy numbers. (2) There are no base mutations between the repeat unit connection regions, which is suitable for the creation of repeat sequences in coding genes. (3) This method is suitable for the creation of 20-100bp short tandem repeats of any coding gene cloned on a plasmid, providing a new research tool for elucidating the relationship between repeat sequences and the pathogenesis of cell senescence, tumorigenesis, and neurodegenerative diseases and for developing related treatments. Summary of the Invention

[0007] The object of the present invention is to provide a method for generating short tandem repeats on the coding gene of a recombinant plasmid by in vitro amplification.

[0008] To achieve the above object, the present invention adopts the following technical means:

[0009] A method for generating short tandem repeats on the coding gene of a recombinant plasmid by in vitro amplification according to the present invention includes the following steps:

[0010] (1) Construct a recombinant plasmid containing the target coding gene as the starting template for in vitro PCR amplification;

[0011] (2) First, use a single-stranded starting primer of a semi-repeat unit to guide the initial PCR amplification of the recombinant plasmid, and then use the product of the initial PCR amplification as a template to perform subsequent PCR amplification through a pair of dual-copy complementary primer pairs. Among them, the short fragment, that is, the repeat unit, has a length of 20-100bp. The forward and reverse primers in the dual-copy complementary primer pair each contain two copies of the short fragment repeat unit. The single-stranded starting primer of the semi-repeat unit is a single-stranded primer that starts from the middle of the forward sequence of the short fragment and terminates at the 3' end of the forward sequence of the short fragment;

[0012] (3) Use the restriction enzyme DpnI to digest the PCR product to remove the starting recombinant plasmid template;

[0013] (4) Transform the digested product into Escherichia coli competent cells;

[0014] (5) Inoculate the colonies into a medium for large-scale culture;

[0015] (6) Identification by bacterial liquid PCR and DNA sequencing by Sanger method.

[0016] Preferably, the recombinant plasmid is pcDNA3.1(-) containing any coding gene.

[0017] Preferably, the two copies of short fragment repeat units in the double-copy complementary primer pair are wild-type sequences or sequences containing mutation sites.

[0018] Preferably, the coding gene is a pathogenic gene related to neurodegenerative diseases.

[0019] Preferably, the neurodegenerative diseases include Huntington's disease, myotonic dystrophy, and fragile X chromosome syndrome.

[0020] In a specific embodiment of the present invention, in order to study the role of phospholipase GPLD1 in anti-aging, based on the recombinant plasmid pcDNA3.1(-)-GPLD1, a repetitive sequence was prepared for the 31bp fragment of the phospholipase active region of its coding gene GPLD1, and a short tandem repeat sequence that originally did not exist in the coding region of GPLD1 was successfully prepared. The specific steps are as follows:

[0021] (1) Construct a recombinant plasmid containing the target coding gene;

[0022] The anti-aging gene GPLD1 was cloned into the pcDNA3.1(-) vector to obtain the recombinant plasmid pcDNA3.1(-)-GPLD1, which was used as the starting template for in vitro PCR amplification.

[0023] (2) DF-62 and DR-62 are fully complementary primers designed with the 31bp short fragment of the GPLD1 phospholipase active region as the template, each containing two copies of the 31bp short fragment repeat unit. In order to facilitate subsequent sequencing identification, GGA in DF-62 was mutated to AAA, and TCC in DR-62 was mutated to TTT. Short-15 is a single-stranded starting primer with a length of 15bp that starts from the middle of the forward sequence of this short fragment and terminates at the 3' end of the forward sequence of this short fragment. Compared with the wild type, the single-stranded starting primer Short-15 of the semi-repeat unit has two mutation sites AA for facilitating the analysis of sequencing results. Seq-F and SR-31 are paired for the identification of bacterial liquid PCR of transformed clones, and Seq-F is also used as the sequencing primer for DNA sequencing by the Sanger method. The specific primer sequences are as follows:

[0024]

[0025] First, initiate the PCR amplification of the pcDNA3.1(-)-GPLD1 recombinant plasmid using the single-stranded starting primer Short-15 of a semi-repeating unit, and then perform subsequent PCR amplification using the double-copy complementary primer pair DF-62 and DR-62 with the starting PCR amplification product as the template;

[0026] (3) Digest the PCR mixture with Dpn I to remove the starting recombinant plasmid template;

[0027] (4) Transform competent Escherichia coli TOP10 cells;

[0028] (5) Inoculate the colonies into the medium for expanded culture;

[0029] (6) Identify by colony PCR and perform DNA sequencing by the Sanger method.

[0030] This repeat sequence has the following characteristics: (1) The directions of the repeating units are the same and the copy numbers are variable. (2) There are no base mutations between the connecting regions of the repeating units, which is suitable for creating repeat sequences in coding genes. (3) This method is suitable for creating short tandem repeat sequences of 20-100 bp of any coding gene cloned on a plasmid.

[0031] The key to this method is to first initiate the PCR amplification of the recombinant plasmid using a single-stranded starting primer of a semi-repeating unit, and then perform subsequent PCR amplification of the recombinant plasmid using a pair of double-copy complementary primer pairs. First, perform 15 cycles of in vitro PCR amplification of the recombinant plasmid pcDNA3.1(-)-GPLD1 using the single-stranded starting primer Short-15 of a semi-repeating unit to generate single-stranded plasmid DNA with partial repeat unit sequences at both the 5' and 3' ends, and then add the double-copy complementary primer pair DF-62 and DR-62 for subsequent 30-cycle PCR amplification (see the schematic diagram of the generation process in Figure 1 Figures A and 1B). After digesting the PCR mixture with Dpn I, transform Escherichia coli TOP10. After selecting clones for inoculation and identification by colony PCR (the identification primers are Seq-F and SR-31, see Table 1 in detail. See the representative agarose gel electrophoresis identification diagram in Figure 2 ), send the bacterial clones with multiple bands for Sanger sequencing. This double-copy complementary primer pair-guided method successfully prepared a short tandem repeat sequence with this 31-bp fragment as the repeating unit. The positive clone ratio generated by this double-copy complementary primer pair-guided method is approximately 40% ( Figure 3 ) and the copy numbers of the repeating units are variable (2-23 copies), with the highest positive clone ratio (33.3%) for those containing 11-15 copies ( Figure 3 Figure B). The connecting regions between the repeating units created by this method are all blunt-end connections, and there are no base mutations between the connecting regions. Figure 4What A shows is the Sanger sequencing result of a positive clone containing 23 copies of short tandem repeats.

[0032] To verify the universality of the dual-copy complementary primer-guided method we established, we used another recombinant plasmid pcDNA3.1(-)-INPP4B as the amplification template, attempting to generate short tandem repeats in the phosphatase activity region of the tumor suppressor gene INPP4B. We designed a pair of dual-copy complementary primer pairs (named DF-58 and DR-58) and a single-stranded starting primer with a length of 14 bp (named Short-14) that starts from the middle of the DF-58 primer and terminates at the 3' end of the DF-58 primer. First, the single-stranded starting primer Short-14 was used to guide the in vitro PCR amplification of the recombinant plasmid pcDNA3.1(-)-3xFlag-INPP4B for 15 cycles to generate single-stranded plasmid DNA with partial repeat unit sequences at both the 5' and 3' ends. Then, the dual-copy complementary primer pairs DF-58 and DR-58 were added for subsequent 30-cycle PCR amplification. After Dpn I digestion of the PCR mixture, it was transformed into Escherichia coli TOP10. After selecting clones for inoculation culture and colony PCR identification, Sanger DNA sequencing was performed. Ten clones were sent for sequencing, and it was found that the INPP4B gene in 4 clones contained short tandem repeats. One representative sequence is shown in Figure 4 Figure B. Note that the directions of the repeat units in this clone are consistent and there are no base mutations in the junction region. This result indicates that the dual-copy complementary primer-guided method we established is universal and suitable for generating short tandem repeats on any coding gene on the recombinant plasmid. The specific steps are as follows:

[0033] (1) Construct a recombinant plasmid containing the target coding gene;

[0034] The tumor suppressor gene INPP4B was cloned into the pcDNA3.1(-) vector to obtain the recombinant plasmid pcDNA3.1(-)-INPP4B, which was used as the starting template for in vitro PCR amplification;

[0035] (2) DF-58 and DR-58 are fully complementary primers designed based on the 29-bp short fragment in the phosphatase activity region of INPP4B, each containing two copies of the 29-bp short fragment repeat unit; Short-14 is a single-stranded starting primer with a length of 14 bp that starts from the middle of the forward sequence of this short fragment and terminates at the 3' end of the forward sequence of this short fragment; Seq-F2 pairs with SR-29 for colony PCR identification of the transformed clones, and Seq-F2 is also used as the sequencing primer for Sanger DNA sequencing; The specific primer sequences are as follows:

[0036]

[0037] First, the initial PCR amplification of the pcDNA3.1(-)-INPP4B recombinant plasmid was initiated with the single-stranded starting primer Short-14 of the semi-repeating unit, and then the subsequent PCR amplification was carried out using the product of the initial PCR amplification as a template with the double-copy complementary primer pair DF-58 and DR-58.

[0038] (3) Digest the PCR mixture with Dpn I to remove the template of the initial recombinant plasmid.

[0039] (4) Transform the competent cells of Escherichia coli TOP10.

[0040] (5) Inoculate the colonies into the medium for large-scale culture.

[0041] (6) Identify the bacterial liquid by PCR and perform DNA sequencing by the Sanger method.

[0042] In summary, using the pcDNA3.1(-)-GPLD1 recombinant plasmid as a template, we successfully created a tandem repeat sequence of a 31-bp fragment in the phospholipase activity region of the GPLD1 coding gene in vitro by the double-copy complementary primer pair-guided method, and verified the universality of this method on the recombinant plasmid pcDNA3.1(-)-INPP4B.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0044] The method of the present invention is based on the in vitro amplification of recombinant plasmids by PCR, which is simple and easy to perform, and has a high positive rate (about 40%). It can achieve the establishment of short fragment tandem repeat sequences in any local 20-100 bp region of any coding gene in vitro. The generated tandem repeat sequences have the following characteristics: (1) The directions of the repeat units are consistent and the copy numbers are variable. (2) There are no base mutations between the connection regions of the repeat units, and the reading frame of the coding gene is not changed.

[0045] The method of the present invention is suitable for generating short fragment tandem repeat sequences on any coding gene of a recombinant plasmid, providing a new research tool for clarifying the relationship between tandem repeat sequences and the pathogenesis of cell senescence, tumorigenesis and various neurodegenerative diseases, and having great economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a schematic diagram for creating a short fragment tandem repeat sequence with variable copy numbers using the 31 bp in the GPLD1 phospholipase activity region of the recombinant plasmid pcDNA3.1(-)-GPLD1 as the repeat unit;

[0047] Among them, (A) Schematic diagram of the structure of recombinant plasmid pcDNA3.1(-)-GPLD1; the left side shows the schematic diagram of the recombinant plasmid, and the right side shows the schematic diagrams of the repeat unit and the generated tandem repeat unit structure. The names, positions, restriction enzyme sites, ampicillin, and neomycin resistance genes of the repeat unit and the primers used are marked at the corresponding positions of the recombinant plasmid with different shapes and shadows; (B) Schematic diagram of generating variable copy number short tandem repeat sequences by the double-copy complementary primer pair-guided method; first, the recombinant plasmid is subjected to initial PCR amplification for 15 cycles with the semi-repeat unit single-stranded starting primer Short-15 primer, and then the complementary primer pair DF-62 and DR-62 is added for subsequent 30-cycle PCR amplification. After the PCR product is digested with Dpn I enzyme, it is transformed into Escherichia coli TOP10. The transformed colonies are identified by colony PCR and sequenced by the Sanger method to determine that the short tandem repeat sequence is successfully prepared on the recombinant plasmid;

[0048] Figure 2 Agarose gel electrophoresis map for identifying short tandem repeat sequences generated by colony PCR of cloned colonies;

[0049] Among them, the M lane is the DNA molecular weight marker, lanes 1, 3, 5, 6, 7, and 8 are single-copy clones, and lanes 2, 4, 9, and 10 are clones containing multiple-copy tandem repeat sequences; among them, the clones corresponding to lanes 9 and 10 are identified by Sanger sequencing and are positive clones containing 23-copy tandem repeat sequences;

[0050] Figure 3 Characteristics of short tandem repeats prepared by the double-copy complementary primer pair-guided method;

[0051] Among them, (A) The ratio of positive clones of short tandem repeat sequences is the result statistically obtained by identifying 100 recombinant plasmid clone colonies by colony PCR; the tandem repeat sequence refers to a sequence containing 2 or more copies of the repeat unit; (B) It is the result summary of the copy number variation analysis of 100 recombinant plasmid clones after Sanger sequencing;

[0052] Figure 4 Sanger DNA sequencing results display of short tandem repeat sequences prepared by the double-copy complementary primer pair-guided method on two genes, the anti-aging gene GPLD1 and the tumor suppressor gene INPP4B;

[0053] Among them, the (A) sequence is a 23-copy short fragment tandem repeat sequence prepared on the GPLD1 gene by the double-copy complementary primer pair-guided method. Among them, 22 ligation regions are all blunt-end ligations, and there are no base mutations between the ligation regions. The shaded regions at both ends of the sequence are non-repetitive sequences adjacent to the repeat unit; the (B) sequence is an 8-copy short fragment tandem repeat sequence prepared on the INPP4B gene by the double-copy complementary primer pair-guided method. Among them, 7 ligation regions are all blunt-end ligations, and there are no base mutations between the ligation regions; the shaded regions at both ends of the sequence are non-repetitive sequences adjacent to the repeat unit. Detailed implementation manners

[0054] The present invention will be further described below in combination with specific implementation examples. The advantages and characteristics of the present invention will become clearer with the experimental description. This implementation case is exemplary and does not constitute any limitation to the scope of the present invention. Those skilled in the relevant art should understand that without departing from the spirit and scope of the present invention, modifications and substitutions can be made to the details and forms of the technical solutions of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

[0055] Example 1: Preparation of a 31-bp short fragment tandem repeat sequence in the phospholipase active region of the anti-aging gene GPLD1 by the double-copy complementary primer pair-guided method

[0056] Materials and methods:

[0057] Prokaryotic cells: Escherichia coli competent TOP10, purchased from Invitrogen.

[0058] Main biochemical reagents:

[0059] PCR-related reagents:

[0060] Taq DNA polymerase and PCR reaction mixture: purchased from Beijing Zhuangmeng Biogene Technology Co., Ltd. Primer synthesis: Anhui General Biology Co., Ltd.

[0061] PCR amplifier: GeneAmp PCR system 9700

[0062] DNA concentration measuring instrument: Nanodrop

[0063] 37°C water bath: Tianjin Taixin Special Instrument Co., Ltd.

[0064] Dpn I restriction endonuclease: NEB, USA

[0065] LB broth and agar: Beijing Aoboxing Biotechnology Co., Ltd.

[0066] Ampicillin: Beijing Saiwen Innovation Technology Co., Ltd.

[0067] 37℃ Incubator: Thermo Company

[0068] Laminar Flow Hood: LABCONCO Company

[0069] Agarose Gel Electrophoresis System: BIO-RAD, USA

[0070] Gel Imaging Scanner: BIO-RAD, USA

[0071] Sanger DNA Sequencing: Completed by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0072] DNA Sequence Analysis Software: SnapGene (SnapGene Inc., California, USA)

[0073] Statistical Data Analysis Software: SPSS 17.0 (SPSS Inc., Chicago, USA)

[0074] Recombinant Plasmid: pcDNA3.1(-)-GPLD1, prepared in the laboratory

[0075] Schematic diagram of creating variable copy number short tandem repeat sequences with 31bp GPLD1 phospholipase activity region of recombinant plasmid pcDNA3.1(-)-GPLD1 as a repeat unit is as Figure 1 shown.

[0076] (1) Primer Design

[0077] DF-62 and DR-62 are fully complementary primers designed based on the 31bp short fragment of the GPLD1 phospholipase activity region, each containing two copies of the 31bp short fragment repeat unit. To facilitate subsequent sequencing identification, GGA in DF-62 was mutated to AAA, and TCC in DR-62 was mutated to TTT. Short-15 is a single-stranded starting primer of a 15bp semi-repeat unit that starts from the middle of the forward sequence of this short fragment and terminates at the 3' end of the forward sequence of this short fragment. Compared with the wild type, there are two mutation sites AA in the single-stranded starting primer of the semi-repeat unit Short-15 to facilitate the analysis of sequencing results. These base mutations do not affect the subsequent functional research of GPLD1. For other general research, the wild-type sequence of the target gene can be directly used. Seq-F is paired with SR-31 for colony PCR identification of transformed clones. Seq-F is also used as the sequencing primer for Sanger DNA sequencing. The specific primer sequences are shown in Table 1:

[0078] Table 1: Primer Sequence Table for Creating 31bp Short Tandem Repeat Sequences of the Anti-aging Gene GPLD1 Phospholipase Activity Region in Vitro

[0079]

[0080] (2) The single-stranded starting primer of the semi-repeating unit, Short-15, guides the starting PCR amplification of the pcDNA3.1(-)-GPLD1 recombinant plasmid.

[0081] 2.1 PCR reaction system:

[0082]

[0083]

[0084] 2.2 Starting PCR reaction running conditions:

[0085] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 8 min, and the number of cycles is 15.

[0086] (3) The double-copy complementary primer pair guides the subsequent PCR amplification of the pcDNA3.1(-)-GPLD1 recombinant plasmid.

[0087] 3.1 PCR reaction system:

[0088]

[0089] 3.2 Subsequent PCR reaction running conditions:

[0090] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 8 min, and the number of cycles is 30.

[0091] (4) Digest the PCR mixture with Dpn I. Add samples to a 1.5-ml centrifuge tube according to the following ratio, mix well and centrifuge, and incubate overnight at 37°C in a water bath for digestion.

[0092]

[0093] (5) Transform Escherichia coli TOP10 competent cells

[0094] 5.1 Take out the TOP10 competent cells from the -80°C refrigerator and the above 10 μl of the Dnp I digestion reaction mixture, and place them on ice for 10 min respectively.

[0095] 5.2 Add 10 μl of the Dnp I digestion reaction mixture to 50 μl of competent cells, gently flick to mix evenly, and then let it stand on ice for 5 min.

[0096] 5.3 Place the mixture in a pre-prepared 42°C water bath and perform heat shock for 55 s.

[0097] 5.4 Let the heat-shocked mixture stand on ice for 3 min.

[0098] 5.5 Add 1 mL of LB broth to the mixture and incubate it in a shaking incubator at 37 °C for 1 h.

[0099] 5.6 Take 200 μL of the above culture and spread it evenly on an LB agar plate containing ampicillin resistance. Place it upside down in an incubator at 37 °C overnight.

[0100] (6) Colony inoculation and identification by colony PCR.

[0101] 6.1 Add 5 mL of LB broth containing ampicillin (100 μg / mL) solution to a 10 mL centrifuge tube. Inoculate a monoclonal colony into it, keep the air unobstructed, and incubate it in a shaking incubator at 37 °C overnight.

[0102] 6.2 The next morning, take 2 μL of the LB bacterial solution and place it in a PCR reaction tube. After placing it in a metal bath at 95 °C for 10 min, prepare the colony PCR reaction system according to the following ratio.

[0103]

[0104] 6.3 Colony PCR reaction running conditions:

[0105] Pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s. The number of cycles is 25.

[0106] 6.4 Identify the colony PCR products by 2% agarose TBE gel electrophoresis.

[0107] Weigh 2 g of agarose, dissolve it in 100 mL of 0.5x TBE, heat it in a microwave oven to dissolve, and after it cools slightly, add 10 μL of nucleic acid dye superRed (10000x), mix well, and pour it into the gel rack. Let it cool and solidify naturally for later use.

[0108] 6.5 Take 8 μL of DL2000 DNA marker and 8 μL of colony PCR product per tube respectively, inject them into the gel loading wells, and electrophorese at a constant voltage of 80 V for 40 min. Then observe the PCR product bands on a BOI-RED gel imaging scanner and scan and save the images. The length of the PCR product of the recombinant plasmid containing a single copy of the repeat unit is 420 bp. The PCR products of the recombinant plasmid containing multiple copies of the repeat unit show ladder-shaped bands with gradually increasing molecular weights. The results are as Figure 2 shown.

[0109] (7) Sanger DNA sequencing.

[0110] Select positive clones with multiple bands in colony PCR, take 2 mL of fresh bacterial solution from each and send them for inspection. The sequencing primer used is Seq-F.

[0111] (8) Analysis of Sanger DNA sequencing results.

[0112] We used SnapGene software to analyze the sequencing results, paying particular attention to the copy number of the repeat units and whether there were nucleotide mutations between the repeat unit junctions. Analyze and summarize the positive ratio of the repeated sequences, the changes in the copy number of the repeat units, and conduct a comparison of statistically significant differences. The results are as Figure 3 shown.

[0113] Figure 4 A shows the Sanger DNA sequencing results of the short tandem repeat sequences prepared by the dual-copy complementary primer pair-guided method on the anti-aging gene GPLD1.

[0114] Example 2: Preparation of a 29-bp short tandem repeat sequence in the phosphatase activity region of the tumor suppressor gene INPP4B by the dual-copy complementary primer-guided method.

[0115] Materials and methods:

[0116] Prokaryotic cells: Escherichia coli competent TOP10, purchased from Invitrogen.

[0117] Main biochemical reagents:

[0118] PCR-related reagents:

[0119] Taq DNA polymerase and PCR reaction mixture: purchased from Beijing Zhuangmeng Biogene Technology Co., Ltd. Primer synthesis: Anhui General Biology Co., Ltd.

[0120] PCR amplifier: GeneAmp PCR system 9700

[0121] DNA concentration measuring instrument: Nanodrop

[0122] 37°C water bath: Tianjin Taixin Special Instrument Co., Ltd.

[0123] Dpn I restriction endonuclease: NEB, USA

[0124] LB broth and agar: Beijing Aoboxing Biotechnology Co., Ltd.

[0125] Ampicillin: Beijing Saiwen Innovation Technology Co., Ltd.

[0126] 37°C incubation incubator: Thermo

[0127] Laminar flow hood: LABCONCO

[0128] Agarose gel electrophoresis system: BIO-RAD, USA

[0129] Gel imaging scanner: BIO-RAD, USA

[0130] Sanger DNA Sequencing: Completed by Beijing Ruibo Xingke Biotechnology Co., Ltd.

[0131] DNA Sequence Analysis Software: SnapGene (SnapGene Inc., California, USA)

[0132] Statistical Data Analysis Software: SPSS 17.0 (SPSS Inc., Chicago, USA)

[0133] Recombinant Plasmid: pcDNA3.1(-)-INPP4B, Prepared in the Laboratory

[0134] (1) Primer Design

[0135] DF-58 and DR-58 are fully complementary primers designed based on the 29bp short fragment repeat unit in the phosphatase active region of INPP4B, each containing two copies of the 29bp short fragment repeat unit. Short-14 is a single-stranded starting primer with a length of 14bp that starts from the middle of the forward sequence of this short fragment and terminates at the 3' end of the forward sequence of this short fragment. Seq-F2 pairs with SR-29 for the identification of bacterial liquid PCR of transformed clones. Seq-F2 is also used as the sequencing primer for Sanger DNA sequencing. The specific primer sequences are shown in Table 2:

[0136] Table 2: Primer Sequence Table for Creating 29bp Short Fragment Tandem Repeat Sequence in the Phosphatase Active Region of Tumor Suppressor Gene INPP4B In Vitro

[0137]

[0138] (2) The single-stranded starting primer Short-14 of the semi-repeat unit guides the initial PCR amplification of the pcDNA3.1(-)-INPP4B recombinant plasmid.

[0139] 2.1 PCR Reaction System:

[0140]

[0141] 2.2 Initial PCR Reaction Running Conditions:

[0142] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 8 min, and the number of cycles is 15.

[0143] (3) The subsequent PCR amplification of the pcDNA3.1(-)-INPP4B recombinant plasmid is guided by the double-copy complementary primer pair.

[0144] 3.1 PCR Reaction System:

[0145]

[0146] 3.2 Follow-up PCR reaction running conditions:

[0147] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 62°C for 30 s, extension at 72°C for 8 min, and the number of cycles is 30.

[0148] (4) Digest the PCR mixture with Dpn I. Add samples to a 1.5-ml centrifuge tube according to the following ratio, mix well and centrifuge, and digest overnight at 37°C in a water bath.

[0149]

[0150] (5) Transform Escherichia coli TOP10 competent cells

[0151] 5.1 Take out the TOP10 competent cells from the -80°C refrigerator and the above 10 μl of Dnp I digestion reaction mixture, and place them on ice for 10 min respectively.

[0152] 5.2 Add 10 μl of the Dnp I digestion reaction mixture to 50 μl of competent cells, gently flick to mix, and then let it stand on ice for 5 min.

[0153] 5.3 Place the mixture in a pre-prepared 42°C water bath and perform heat shock for 55 s.

[0154] 5.4 Let the heat-shocked mixture stand on ice for 3 min.

[0155] 5.5 Add 1 ml of LB broth to the mixture and culture it in a 37°C constant temperature shaker for 1 h.

[0156] 5.6 Take 200 μL of the above culture and spread it evenly on an LB agar plate containing ampicillin resistance, and place it upside down in a 37°C incubator overnight.

[0157] (6) Colony inoculation and colony PCR identification

[0158] 6.1 Add 5 ml of LB broth ampicillin (100 μg / mL) solution to a 10-ml centrifuge tube. Inoculate a monoclonal colony into it, keep the air unobstructed, and culture it overnight in a 37°C constant temperature shaker.

[0159] 6.2 The next morning, take 2 μL of the LB bacterial solution and place it in a PCR reaction tube. After placing it in a 95°C metal bath for 10 min, prepare the bacterial solution PCR reaction system according to the following ratio.

[0160]

[0161] 6.3 Bacterial solution PCR reaction running conditions

[0162] Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, and the number of cycles was 25.

[0163] The PCR products of the bacterial liquid were identified by 6.42% agarose TBE gel electrophoresis.

[0164] Weigh 2 g of agarose, dissolve it in 100 ml of 0.5x TBE, heat it in a microwave oven until dissolved, add 10 μl of nucleic acid dye superRed (10000x) after cooling slightly, and mix well. Then pour it into the gel rack. Let it cool and solidify naturally for later use.

[0165] 6.5 Take 8 μL of DL2000 DNA marker and 8 μL of the PCR products of the bacterial liquid per tube, inject them into the gel loading wells, and perform electrophoresis at a constant voltage of 80 V for 40 min. Then observe the PCR product bands on a BOI-RED gel imaging scanner and scan and save the images. The length of the PCR product of the recombinant plasmid containing a single-copy repeat unit is 509 bp. The PCR products of the recombinant plasmid containing multiple-copy repeat units show ladder-shaped bands with gradually increasing molecular weights.

[0166] (7) DNA sequencing by the Sanger method.

[0167] Select positive clones with multiple bands in the PCR of the bacterial liquid, take 2 ml of fresh bacterial liquid for each and send them for inspection. The sequencing primer used is Seq-F2.

[0168] (8) Analysis of the DNA sequencing results by the Sanger method.

[0169] We used SnapGene software to analyze the sequencing results, paying special attention to the copy number of the repeat units and whether there are base mutations between the repeat unit junction regions. Analyze and summarize the positive ratio of the repeated sequences, the changes in the copy number of the repeat units, and perform a comparison of statistically significant differences.

[0170] Figure 4 Figure B shows the DNA sequencing results by the Sanger method of the short tandem repeat sequences prepared by the double-copy complementary primer pair-guided method on the tumor suppressor gene INPP4B.

Claims

1. A method for generating a tandem repeat sequence of a 31bp short fragment in the phospholipase active region of the anti-aging gene GPLD1 by in vitro amplification of a recombinant plasmid, characterized in that, The method described above includes the following steps: (1) Construct a recombinant plasmid containing the target coding gene; Clone the anti-aging gene GPLD1 into the pcDNA3.1(-) vector to obtain the recombinant plasmid pcDNA3.1(-)-GPLD1, which serves as the starting template for in vitro PCR amplification; (2) DF-62 and DR-62 are fully complementary primers designed based on the 31-bp short fragment of the GPLD1 phospholipase active region. Each contains two copies of the 31-bp short fragment repeat unit. To facilitate subsequent sequencing identification, GGA in DF-62 is mutated to AAA, and TCC in DR-62 is mutated to TTT. Short-15 is a single-stranded starting primer with a length of 15 bp that starts from the middle of the forward sequence of this short fragment and terminates at the 3' end of the forward sequence of this short fragment. Compared with the wild type, the single-stranded starting primer Short-15 for the semi-repeat unit has two mutation sites AA to facilitate the analysis of sequencing results. Seq-F pairs with SR-31 for colony PCR identification of transformed clones, and Seq-F also serves as the sequencing primer for Sanger DNA sequencing. The specific primer sequences are as follows: Short 15: 5’-ATAGTCTGGGAATTG-3’ 5’-GCAGATGTCAGCTAAAATAGTCTGGGCCTTGGC DF-62: AGATGTCAGCTAAAATAGTCTGGGCCTTG-3’ 5’-CAAGGCCCAGACTATTTTAGCTGACATCTGCCA DR-62: AGGCCCAGACTATTTTAGCTGACATCTGC-3’ SR-31: 5’-CAAGGCCCAGACTATTCCAGCTGACATCTGC-3’ Seq-F: 5’-AATTGGTACCATGTCTGCTTTC-3’ First, use the single-stranded starting primer Short-15 for the semi-repeat unit to guide the initial PCR amplification of the pcDNA3.1(-)-GPLD1 recombinant plasmid, and then use the product of the initial PCR amplification as the template to perform subsequent PCR amplification with the double-copy complementary primer pair DF-62 and DR-62; (3) Digest the PCR mixture with Dpn I enzyme to remove the starting recombinant plasmid template; (4) Transform Escherichia coli TOP10 competent cells; (5) Inoculate the colonies into the medium for large-scale culture; (6) Perform colony PCR identification and Sanger DNA sequencing.

2. A method for generating a tandem repeat sequence of a 29-bp short fragment of the phosphatase activity region of the tumor suppressor gene INPP4B by in vitro amplification of a recombinant plasmid, characterized in that, It includes the following steps: (1) Construct a recombinant plasmid containing the target coding gene; Clone the tumor suppressor gene INPP4B into the pcDNA3.1(-) vector to obtain the recombinant plasmid pcDNA3.1(-)-INPP4B, which serves as the starting template for in vitro PCR amplification; (2) DF-58 and DR-58 are fully complementary primers designed based on a 29-bp short fragment in the phosphatase activity region of INPP4B, each containing two copies of the 29-bp short fragment repeat unit; Short-14 is a single-stranded starting primer with a length of 14 bp that starts from the middle of the forward sequence of this short fragment and ends at the 3' end of the forward sequence of this short fragment; Seq-F2 pairs with SR-29 for colony PCR identification of transformed clones, and Seq-F2 is also used as the sequencing primer for Sanger DNA sequencing; the specific primer sequences are as follows: Short 14: 5’-AGCCTGAGAACTTC-3’ DF-58: 5’-CAATTGCTGGGGCTGAGCCTGAGAACTTCCAATTGCTGG GGCTGAGCCTGAGAACTTC-3’ DR-58: 5’-GAAGTTCTCAGGCTCAGCCCCAGCAATTGGAAGTTCTCAGGCTCAGCCCCAGCAATTG-3’ SR-29: 5’-GAAGTTCTCAGGCTCAGCCCCAGCAATTG-3’ Seq-F2: 5’-ATGGAAATTAAAGAGGAAGGGGCATC-3’ First, use the single-stranded starting primer Short-14 of the semi-repeated unit to guide the initial PCR amplification of the pcDNA3.1(-)-INPP4B recombinant plasmid, and then use the product of the initial PCR amplification as a template to perform subsequent PCR amplification with the double-copy complementary primer pair DF-58 and DR-58; (3) Digest the PCR mixture with Dpn I to remove the initial recombinant plasmid template; (4) Transform Escherichia coli TOP10 competent cells; (5) Inoculate the colonies into the medium for expanded culture; (6) Perform colony PCR identification and Sanger DNA sequencing.

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