A kind of construction method of tale repeat sequence vector
A repeating sequence and vector technology, applied in the field of molecular biology, can solve problems such as large workload, unfavorable TALE technology promotion, complexity, etc., and achieve the effect of improving work efficiency, reducing the number of vectors and workload, and simple operation.
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Embodiment 1
[0043] Example 1 A one-step approach to building TALENs
[0044] 1. According to the TALEN site selection principle, select a 16bpTALE binding site in the exon region of the gene.
[0045] 2. Use the dimer amplification primer (shown in Table 1) to PCR amplify the TALE dimer at the corresponding position of the TALE binding site, and purify and recover the PCR product.
[0046] 3. Select 10 ng of each dimer PCR recovery fragment at the corresponding position, 100 ng of TALEN backbone vector, add BsaI, T4 / T7 DNA ligase, CutSmart buffer, ATP, etc. and mix well, and perform the following reaction operation in the PCR machine: 37 ℃ reaction 5 minutes at 20°C for 5 minutes, 20 cycles; 5 minutes at 50°C and 5 minutes at 80°C.
[0047] 4. After the reaction, the product was added to the competent cells for transformation. The next day, single clones were picked and shaken, and positive clones were identified by colony PCR.
[0048] Table 1 Dimer amplification primers
[0049] ...
Embodiment 2
[0051] Example 2 Construction of TALE dimer starting plasmid
[0052] Due to the nature of TALE repeats, in order to enable PCR to successfully amplify TALE dimers, the inventors optimized the DNA sequences encoding the two TALE monomers before and after using the degeneracy of codons, and at the same time the second single The penultimate amino acid of the body is changed from A to D. First, the first and second TALE monomers were PCR amplified with Q5 DNA polymerase using primers F9 / R9 and F10 / R10, respectively, using the nucleotide sequence comprising SEQ ID NO: 2 as a template, followed by primers F9 / R10 The first and second TALE monomers were spliced together by overlapping PCR; then double digested with EcoRI / BamHI, the TALE dimer unit was cloned into the pEGFP-N1 vector, and the plasmid was extracted after sequencing.
[0053] SEQ ID NO: 2CTCACCCCAGAGCAGGTCGTGGCAATTGCGAGCAACATCGGGGGAAAGCAGGCACTCGAAACCGTCCAGAGGTTGCTGCCTGTGCTGTGCCAAGCGCACGGATTGACGCCCGCACAAGTAGTTGCAAT...
Embodiment 3
[0058] Example 3 TALE one-step construction of TALEN vector
[0059] First, verify whether PCR can amplify the TALE dimer band. PCR amplification was performed using the amplification primers shown in Table 1, and the electrophoresis bands showed that the corresponding 1-8 dimers could be amplified ( image 3 ). The PCR products were then recovered and verified using the method in Example 1 to obtain correctly assembled positive clones. image 3 In b and c, T4 or T7 DNA ligases were used to ligate, respectively, and it was found that the correctly assembled TALEN vector could be obtained with these two enzymes.
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