CN110358812B - Primer and detection method for gene knockout and recombination positive clone detection of lambda-Red recombination system - Google Patents
Primer and detection method for gene knockout and recombination positive clone detection of lambda-Red recombination system Download PDFInfo
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Abstract
The invention discloses a primer and a detection method for detecting positive clone of gene knockout recombination of a lambda-Red recombination system. Belongs to the field of biotechnology engineering. The invention provides a universal detection primer, and a design method thereof is that tetracycline is used as a primertetA pair of complementary primers is designed at about 1/3 of the middle of the RA gene, and is combined with the original identification primer for detecting the recombination positivity, so that the primers are respectively used for detecting the 3 'end sequence and the 5' end sequence of the lambda-Red recombination system gene knockout recombination positive clone which takes tetracycline gene tetRA as a replacement gene. The design of the detection primers leads the detection of the recombinant positive clones to have 3 choices, and can completely avoid the difficult problem of difficult judgment of positive results caused by small difference between the sequence to be knocked out and the tetracycline tetRA gene. And the universal primer is used for detecting the recombinant positive clone, so that the correctness of the tetracycline tetRA gene insertion position can be more effectively proved.
Description
Technical Field
The invention belongs to the field of biotechnology engineering, and particularly relates to a primer and a detection method for detecting recombinant positive clones after target genes are knocked out by a lambda-Red recombinant system.
Background
The homologous recombination system based on lambda phage Red recombinase is widely used for the genetic engineering research of Escherichia coli at present. The Red recombination system consists of three proteins: the Exo protein is an exonuclease, is combined at the end of double-stranded DNA, degrades the DNA from a 5' end to a3' end, and generates a3' overhang; beta protein is combined on the single-stranded DNA to mediate the annealing of the complementary single-stranded DNA; the Gam protein can be combined with RecBCD enzyme to inhibit the activity of degrading exogenous DNA. The Red homologous recombination technology has the characteristics of short homologous sequence (40-60 bp) and high recombination efficiency. The technology can perform operations such as gene knockout, knock-in, point mutation and the like on any site of a DNA target molecule without using restriction enzymes and ligases.
The tetracycline resistance gene tetrA is easy to detect as a genetic screening marker and has conditional lethal property, so that the tetracycline resistance gene tetrA is often used as a replacement gene to replace a gene needing knockout or an inserted sequence. The Red homologous recombination technology enables a gene engineering experiment with high difficulty to be smoothly carried out, and greatly promotes the development of functional genome research. The tetracycline resistance tetRA gene is used as a replacement gene for knocking out a target gene, and the size of the tetracycline resistance tetRA gene is 1911bp; the homologous recombination sequence is usually the homologous arm sequence at both sides of the target gene, taking the length of 40bp as an example; the selection position of the identifying primer for identifying whether the gene is recombined is usually the homologous arm outside the homologous recombination sequence, taking the length of 18bp as an example; therefore, the size of the fragment for homologous recombination is 1911+40+ 1991bp; the result of PCR detection of the recombination positive is 1991+18+ 2027bp, and the corresponding negative control sizes are: the size of the original gene x +40+18 +116bp. Because the sizes of the genes to be knocked out are different, if the size difference between the sequence to be knocked out and the fragment of the tetracycline resistance tetRA gene is larger, the positive result is more different from the negative control fragment, and the positive result is easy to identify; if the size difference between the gene sequence to be knocked out and the tetracycline resistance tetRA gene fragment is small, a positive result and a negative control are difficult to identify due to the resolution problem of agarose gel electrophoresis, and the result cannot be accurately judged.
Disclosure of Invention
In order to overcome the defects of the existing detection method, the invention aims to provide a universal detection primer, and the design method is to design a pair of complementary primers at about 1/3 of the middle of the tetracycline tetRA gene, combine the complementary primers with the original identification primer for detecting the positive recombination and use the complementary primers and the original identification primer for detecting the 3 'end sequence and the 5' end sequence of the lambda-Red recombination system gene knockout recombination positive clone which takes the tetracycline gene tetRA as a replacement gene respectively. The design of the detection primer leads the detection of the recombinant positive clone to have 3 choices, and can completely avoid the difficult problem of difficult judgment of positive results caused by small difference between the sequence to be knocked out and the tetracycline tetRA gene. And the universal primer is used for detecting the recombinant positive clone, so that the correctness of the tetracycline tetRA gene insertion position can be more effectively proved.
The invention also aims to provide a detection method for positive gene knockout and recombination of the lambda-Red recombination system by taking the tetracycline gene tetRA as a replacement gene.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, a method for detecting a lambda-Red recombination system gene knockout recombination positive clone by using a tetracycline resistance gene tetRA as a replacement gene comprises the following steps:
(1) Designing a primer:
obtaining a tetRA gene sequence and a sequence of a gene H to be knocked out from an NCBI database, designing a pair of primers H-tetRAR and H-tetrAF for amplifying homologous recombination sequences, wherein the 5 'end of each primer has 35-60 bp which are respectively an upstream homologous sequence a or a' fragment of the gene H to be knocked out, the sequence lengths of the primers are both L1, the 3 'ends are respectively a primer sequence b or a b' fragment carried by the two sides of a tetracycline resistance gene tetRA, and the sequence length of the tetracycline resistance gene tetRA is L; designing identifying primers JC-HF and JC-HR for identifying whether the genes are recombined, wherein the sequences of the identifying primers are respectively positioned in a c or c 'segment outside an upstream and downstream homologous sequence a or a' segment of a gene H to be knocked out, and the sequence lengths of the primers JC-HF and JC-HR are both L2; designing two complementary universal primers TYJC-tetrA and TYJC-tetrAR, wherein the sequence of the universal primers is a d fragment in a tetracycline resistance gene tetrA, the sequence length from a sequence b to a sequence d on the tetracycline resistance gene tetrA is L3, and the sequence length from a sequence d 'to a sequence b' on the tetracycline resistance gene tetrA is L4;
(2) Amplification of a DNA fragment containing the tetRA gene;
(3) Preparing competent cells;
(4) Electrotransfer and screening and identifying target strains;
detecting the screened target strain by using identifying primers JC-HF and JC-HR, wherein the theoretical length value of a target fragment is = L2+ L1+ L + L1+ L2; detecting the screened target strain by using JC-HR and TYJC-tetrAF, wherein the length of the target fragment is theoretical value = L1+ L2+ L4, and detecting the screened target strain by using JC-HF and TYJC-tetrAR, wherein the length of the target fragment is theoretical value = L1+ L2+ L3;
if the length of a PCR amplified fragment obtained by detecting and screening target strains by using JC-HF and JC-HR, JC-HR and TYJC-tetrAF or JC-HF and TYJC-tetrAR 3 pairs of primers is close to the theoretical length value of the target fragment, the tetrA gene targeting fragment successfully knocks out the gene H to be knocked out.
Preferably, the two complementary universal primers TYJC-tetrA and TYJC-tetrA in step (1) of the above detection method are about 1/3 of the tetracycline resistance gene tetRA from the 5' end, and the sequences are shown in SEQ ID NO:1 and SEQ ID NO: 2.
The schematic diagram of the technical scheme of the invention is shown in figure 1.
Compared with the prior art, the invention has the advantages that:
the detection primer designed by the invention has 3 choices for detecting positive clones, and can completely avoid the problem that the positive result is difficult to judge because the difference between the sequence to be knocked out and the tetracycline tetRA gene is small. And the universal primer is used for detecting positive clones, so that the correctness of the tetracycline tetRA gene insertion position can be more effectively proved.
Drawings
FIG. 1 is a schematic illustration of the technical solution of the present invention;
FIG. 2 is a diagram showing the PCR product gel cutting recovery electrophoresis detection for Tar gene recombination;
m: DL2000 DNA standard; 1: recovering products from tetracycline resistant gene glue.
FIG. 3 shows a PCR identification chart of a colony of a Tar knockout strain;
m: DL2000 DNA standard; lane 1 is the control group Tar gene, the size is 1662bp, lane 2 is the PCR detection result of primers JC-TarR and JC-TarF, and the theoretical value is 2070bp close to the target fragment; lane 3 is a PCR amplified fragment of primers JC-Tar-R and TYJC-tetrAF, which is close to the theoretical value 1469bp of the target fragment; lane 4 is a PCR amplified fragment of primers TYJC-tetRAR and JC-Tar-F, which is close to the theoretical value of 576bp for the fragment of interest.
FIG. 4 is a schematic diagram of the positions of two genes, cheB and CheR, which are adjacent in the genome of E.coli;
FIG. 5 gel cutting recovery electrophoresis detection of PCR products for recombination of CheB and CheR genes;
m: DL2000 DNA standard; 1: recovering products from tetracycline resistant gene glue.
FIG. 6 is a PCR identification chart of a colony of a CheB and CheR gene knockout strain;
m: DL2000 DNA standard; lane 1 was detected with MG1655 original strain control, 2029bp in size, lane 2, 3, 4 were primers JC-CheRF and JC-CheBR for PCR detection of positive clones 2070bp in size; lane 5 is a control panel of MG1655 original strain with primers JC-CheBR and TYJC-tetrAF for PCR detection, no band, and agreement with expectations; lanes 6, 7, 8 are PCR-detected positive clones with 1469bp primers JC-CheBR and TYJC-tetrAF; lane 9 is a control panel with primers TYJC-tetRAR and JC-CheRF for PCR detection of the original strain of MG1655, with no bands, and is expected to be consistent; lanes 10, 11, 12 are PCR positive clones with 576bp size using primers TYJC-tetRAR and JC-CheRF.
Detailed Description
The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.
In the following examples, the tetRA gene sequence was obtained from the NCBI database, with the 18bp sequence of the tetRA gene double underlined, 3' to the PCR amplification primers of the exogenous DNA for tetracycline homologous recombination. The single underline is a universal detection primer used in combination with an identification primer used to identify whether a gene is recombinant.
The universal primer sequences are as follows, and the two primer sequences are complementary:
TYJC-tetrAF:5' ACAATGTAGGCTTGCTCTA3 ' (for detecting 3' sequence)
TYJC-tetAR: 5' TAGAGCAGCCTACATTGT ' (for detecting 5' sequence)
Example 1 detection of Positive clones knocking out the Tar Gene of E.coli Aspartaric acid (aspartic acid) receptor by Using Universal detection primers
(1) Designing a primer:
primers used for homologous recombination are Tar-tetRAR and Tar-tetRAF; the design principle is that the 5 'end is a homologous region (not underlined) of a tar outside gene, the 3' end is a homologous sequence (underlined) of a tetracycline resistance gene tetRA, and primers used for detecting recombinants are JC-TarF and JC-TarR. The sequences of these primers are shown in Table 1 and synthesized by Biotechnology engineering (Shanghai) GmbH.
TABLE 1 primers used in example 1
(2) Amplification of a DNA fragment containing the tetRA gene:
and (3) performing PCR amplification by using DNA of escherichia coli DH10Bac containing tetracycline resistance genes as a template and using Tar-tetRAR and Tar-tetRAF as primers to obtain DNA fragments with two ends respectively homologous with the upstream and downstream of the Tar gene and the middle of the tetracycline resistance genes tetRA. This fragment was recovered on agarose gel for further electrotransformation, as shown in FIG. 2.
(3) Preparation of competent cells:
MG1655 strain containing pKD46 plasmid was cultured overnight at 30 ℃ in LB medium containing 20. Mu.g/mL of Amp, after which it was inoculated in LB medium containing 20. Mu.g/mL of Amp at a ratio of 1.
(4) Electrotransfer and screening and identifying of target strain
Mixing the DNA fragment (100 ng) recovered from the gel with 80 μ L of competent cells in an electric rotating cup, carrying out ice bath for 5-10min, then carrying out electric shock by 2200V, rapidly adding 1000 μ L of LB culture solution after electric shock, carrying out shake culture at 30 ℃ for 30min, then coating a plate on a 20 μ g/mL tetracycline plate, carrying out overnight culture at 37 ℃, further streaking and purifying the plate on the tetracycline plate, and then detecting the result by using a detection primer, wherein the colony grown on the plate is a tetracycline-resistant colony, and the result is shown in figure 3. Lane 1 is the control group Tar gene, the size is 1662bp, lane 2 is the fragment of primer JC-TarR and JC-TarF for PCR amplification, more than 2000bp, and is close to the theoretical value 2070bp of the target fragment; lane 3 is a PCR amplified fragment of primers JC-Tar-R and TYJC-tetrAF, ranging from 1000 to 2000bp, close to the theoretical value 1469bp of the target fragment; lane 4 is a PCR amplified fragment of primers TYJC-tetRAR and JC-Tar-F, which is close to the theoretical value of 576bp for the fragment of interest. Indicating that the tetrA gene targeting fragment has successfully knocked out the Tar gene.
Example 2 detection of Positive clones simultaneously knocking out the genes of the Escherichia coli methyl modification System CheB (methylase) and CheR (methyllipase) Using Universal detection primers
(1) Designing a primer:
as shown in fig. 4, cheB and CheR are two adjacent genes in the genome of escherichia coli, and the sequence numbers of the CheB genes are: NC — 000913.3 (1967452.. 1968501, complete); the sequence number of the CheR gene is NC-000913.3 (1968504.. 1969364, supplement). Therefore, when the two genes are knocked out simultaneously, the size of a target sequence is 1913bp, and primers for homologous recombination are CheB-tetrAR and CheR-tetrAF; the design principle is that the 5 'end is a homologous region (not underlined) of the CheB and CheR lateral genes, the 3' end is a homologous sequence (underlined) of the tetracycline resistance gene tetRA, and primers used for detecting recombinants are JC-CheRF and JC-CheBR. The sequences of these primers are shown in Table 2 and synthesized by Biotechnology engineering (Shanghai) Ltd.
TABLE 2 primers used in example 2
(2) Amplification of a DNA fragment containing the tetRA gene:
and carrying out PCR amplification by taking DNA of escherichia coli DH10Bac containing tetracycline resistance genes as a template and CheB-tetRAR and CheR-tetRAF as primers to obtain a DNA fragment with two ends respectively homologous with the upstream and downstream of the CheB and CheR genes and the middle tetracycline resistance gene tetRA. This fragment was recovered on agarose gel for further electrotransformation, as shown in FIG. 5.
(3) Preparation of competent cells:
MG1655 strain containing pKD46 plasmid was cultured overnight at 30 ℃ in LB medium containing 20. Mu.g/mL of Amp, then inoculated with LB medium containing 20. Mu.g/mL of Amp at a ratio of 1.
(4) Electrotransfer and screening and identifying of target strain
Mixing the DNA fragment (100 ng) recovered from the gel with 80 μ L of competent cells in an electric rotating cup, carrying out ice bath for 5-10min, then carrying out electric shock by 2200V, rapidly adding 1000 μ L of LB culture solution after electric shock, carrying out shake culture at 30 ℃ for 30min, then coating a plate on a 20 μ g/mL tetracycline plate, carrying out overnight culture at 37 ℃, further streaking and purifying the plate on the tetracycline plate, and then detecting the result by using a detection primer, wherein the colony grown on the plate is a tetracycline-resistant colony, and the result is shown in FIG. 6. Lane 1 was a 2029bp PCR-detected positive clone with MG1655 as the original strain control, and lanes 2, 3 and 4 were primers JC-CheRF and JC-CheBR with a size of 2070bp; lane 5 is a control panel of MG1655 original strain PCR-tested with primers JC-CheBR and TYJC-tetrAF, with no bands, consistent with expectations; lanes 6, 7, and 8 are PCR-detected positive clones with primer JC-CheBR and TYJC-tetrAF, size 1469bp; lane 9 is a control panel with primers TYJC-tetRAR and JC-CheRF for PCR detection of the original strain of MG1655, with no bands, and is expected to be consistent; lanes 10, 11, 12 are PCR-detected positive clones with primers TYJC-tetRAR and JC-CheRF, and the size is 576bp. As can be seen from lanes 1 and 2, 3 and 4 in the figure, since the difference between the size of the knocked-out gene and the size of the substituted gene is small, if the original detection primer is used for PCR detection, the resolution of agarose gel electrophoresis cannot identify the difference between the positive strain and the original strain, and thus whether the gene is knocked out cannot be judged; if the universal detection primer is matched, the target genes CheB and CheR can be definitely judged to be successfully replaced by the tetracycline resistance gene tetRA.
Sequence listing
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Claims (1)
1. A detection method of a lambda-Red recombination system gene knockout recombination positive clone by taking a tetracycline resistance gene tetRA as a replacement gene is characterized by comprising the following steps:
(1) Designing a primer:
obtaining a tetRA gene sequence and a sequence of a gene H to be knocked out from an NCBI database, designing a pair of primers H-tetRAR and H-tetRAF for amplifying a homologous recombination sequence, wherein the 5' end of each primer has 35-60 bp and is respectively a fragment of an upstream homologous sequence a or a ' of the gene H to be knocked out, the sequence lengths of the primers are L1, the 3' ends are respectively a primer sequence b or a fragment of a primer sequence b carried by the tetRA of a tetracycline resistance gene, and the sequence length of the tetRA of the tetracycline resistance gene is L; designing identifying primers JC-HF and JC-HR for identifying whether the genes are recombined, wherein the sequences of the identifying primers are respectively positioned in a c or c 'segment outside an upstream and downstream homologous sequence a or a' segment of a gene H to be knocked out, and the sequence lengths of the primers JC-HF and JC-HR are both L2; designing two complementary universal primers TYJC-tetrA and TYJC-tetrAR, wherein the sequence of the universal primers is a d or d ' fragment in a tetracycline resistance gene tetrA, the sequence length from the sequence b to the sequence d on the tetracycline resistance gene tetrA is L3, and the sequence length from the sequence d ' to the sequence b ' on the tetracycline resistance gene tetrA is L4;
the two complementary universal primers TYJC-tetrA and TYJC-tetrAR are arranged at about 1/3 position away from the 5' end of a tetracycline resistance gene tetrA, and the sequences of the two complementary universal primers are shown as SEQ ID NO 1 and SEQ ID NO 2;
(2) Amplification of a DNA fragment containing the tetRA gene;
(3) Preparing competent cells;
(4) Electrotransfer and screening and identifying target strains;
detecting the screened target strain by using identifying primers JC-HF and JC-HR, wherein the length of a target fragment is the theoretical value = L2+ L1+ L + L1+ L2; detecting the screened target strain by using JC-HR and TYJC-tetRAF, wherein the length of a target fragment is the theoretical value = L1+ L2+ L4, and detecting the screened target strain by using JC-HF and TYJC-tetRAR, wherein the length of the target fragment is the theoretical value = L1+ L2+ L3;
if the length of a PCR amplified fragment obtained by detecting the screened target strain by using JC-HF and JC-HR, JC-HR and TYJC-tetrAF or JC-HF and TYJC-tetrAR 3 pairs of primers respectively is close to the theoretical length value of the target fragment, the tetRA gene targeting fragment successfully knocks out the gene H to be knocked out.
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| US7674621B2 (en) * | 2004-05-21 | 2010-03-09 | The United States Of America As Represented By The Department Of Health And Human Services | Plasmids and phages for homologous recombination and methods of use |
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| CN1352694A (en) * | 1998-11-09 | 2002-06-05 | 阿文蒂斯药物股份有限公司 | Novel system for regulating transgene expression |
| CN102952793A (en) * | 2012-10-12 | 2013-03-06 | 上海中科高等研究院 | Genetic recombination method of klebsiella pneumoniae by utilizing short homologous sequence |
| CN105505974A (en) * | 2016-01-21 | 2016-04-20 | 浙江大学 | Method for implementing Escherichia coli gene traceless homologous recombination |
| CN109554323A (en) * | 2018-12-13 | 2019-04-02 | 武汉生物工程学院 | A kind of the gene mutator strain and preparation method and application of the forfeiture of Escherichia coli dnaQ intergenic suppression function |
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