传统的DNA重组方法Type Ⅱ型限制酶技术受到片段纯化的限制,无法做到复杂混合体系中DNA片段的特异性连接。为解决这个问题,本研究将耐热连接酶链式反应(Thermostable ligase chain reaction, TLCR)引入DNA片段的连接与捕获。该技术利用耐热型DNA连接酶的特性,在热循环反应中配合针对目的片段末端序列设计的单链寡核苷酸连接模板——Helper,实现目的片段的特异性连接和产物数量的指数性增长。两个质粒构建实验被用于验证TLCR技术的可行性和应用效果。首先利用TLCR技术从一个未经纯化的含有7种不同大小片段的混杂体系中特异性地将一段1.5 kb的片段捕获进载体,随机抽取的克隆样品经检验准确率达到80%,验证TLCR技术在复杂混合体系中特异性连接DNA片段的可行性和准确性。在另一个质粒构建实验中,TLCR技术从λ噬菌体基因组Hind消化物中将两段总长达27 kb的片段按顺序捕获进载体,随机抽取的克隆样品经检验同样达到了80%的准确率。结果表明,TLCR技术能够简化DNA重组实验的操作,并且适用于多片段和大片段的连接,可以为生物学研究提供便利。
周翔达,宋晓,怀聪,孙海燕,陈红岩,卢大儒
. 耐热DNA连接酶介导的TLCR技术在简化DNA重组实验中的应用[J]. 遗传, 2016
, 38(2)
: 163
-169
.
DOI: 10.16288/j.yczz.15-428
The traditional Type Ⅱ restriction enzyme-based method is restricted by the purification steps, and therefore, cannot be applied to specific DNA assembly in chaotic system. To solve this problem, Thermostable Ligase Chain Reaction (TLCR) was introduced in the process of DNA assembly and capture. This technique combines the feature of thermostable DNA ligase and sequence specific oligo ligation template, “Helper”, to achieve specific assembly of target fragments and exponential increase of products in multiple thermocyclings. Two plasmid construction experiments were carried out in order to test the feasibility and practical performance of TLCR. One was that, TLCR was used to specifically capture a 1.5 kb fragment into vector from an unpurified chaotic system which contained 7 different sizes of fragments. The results showed that the capturing accuracy was around 80%, which proved the feasibility and accuracy of using TLCR to specific assembly of DNA fragments in a complicated mixed system. In the other experiment, TLCR was used to capture two fragments (total length was 27 kb) from Hind Ⅲ digestion of Lambda genome into vector by order. The results also showed an accuracy of around 80%. As demonstrated in the results, TLCR can simplify the process of DNA recombination experiments and is suitable for the assembly of multiple and large DNA fragments. This technique can provide convenience to biological experiments.
[1] Lu Q. Seamless cloning and gene fusion. Trends Biotechnol , 2005, 23(4): 199-207.
[2] Li MZ, Elledge SJ. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods , 2007, 4(3): 251-256.
[3] Peccoud J, Isalan M. The PLOS ONE synthetic biology collection: six years and counting. PLoS One , 2012, 7(8): e43231.
[4] Ellis T, Adie T, Baldwin GS. DNA assembly for synthetic biology: from parts to pathways and beyond. Integr Biol , 2011, 3(2): 109-118.
[5] Yount B, Denison MR, Weiss SR, Baric RS. Systematic assembly of a full-length infectious cDNA of mouse hepatitis virus strain A59. J Virol , 2002, 76(21): 11065-11078.
[6] Lebedenko EN, Birikh KR, Plutalov OV, Berlin YA. Method of artificial DNA splicing by directed ligation (SDL). Nucleic Acids Res , 1991, 19(24): 6757-6761.
[7] Rebatchouk D, Daraselia N, Narita JO. NOMAD: A versatile strategy for in vitro DNA manipulation applied to promoter analysis and vector design. Proc Natl Acad Sci USA , 1996, 93(20): 10891-10896.
[8] Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene , 1989, 77(1): 61-68.
[9] Szybalski W, Kim SC, Hasan N, Podhajska AJ. Class-IIs restriction enzymes-a review. Gene , 1991, 100: 13-26.
[10] Aslanidis C, de Jong PJ. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res , 1990, 18(20): 6069-6074.
[11] Hosfield T, Lu Q. Influence of the amino acid residue downstream of (Asp) 4 Lys on enterokinase cleavage of a fusion protein. Anal Biochem , 1999, 269(1): 10-16.
[12] Wyborski DL, Bauer JC, Zheng CF, Felts K, Vaillancourt P. An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions. Protein Expr Purif , 1999, 16(1): 1-10.
[13] Lu Q, Bauer JC, Greener A. Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins. Gene , 1997, 200(1-2): 135-144.
[14] Hosfield T, Lu Q. S. pombe expression vector with 6x(His) tag for protein purification and potential for ligation-independent cloning. Biotechniques , 1999, 27(1): 58-60.
[15] Zhu B, Cai G, Hall EO, Freeman GJ. In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques , 2007, 43(43): 354-359.
[16] Yu H, Merchant B, Scheffel C, Yandava C, Drazen JM, Huff J. Automated detection of single nucleotide polymorphism in β-2 adrenergic receptor gene using LCx®. Clin Chim Acta , 2001, 308(1-2): 17-24.
[17] Abravaya K, Carrino JJ, Muldoon S, Lee HH. Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Res , 1995, 23(4): 675-682.
