ABSTRACT
In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.
Subject(s)
Clone Cells , Mutagenesis, Site-Directed , Mutation , Plasmids/genetics , Polymerase Chain Reaction/methodsABSTRACT
Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.
Subject(s)
Base Sequence , Cloning, Molecular , Genetic Vectors , Genetics , Mutagenesis, Site-Directed , Methods , Nucleic Acid Amplification Techniques , Plasmids , Polymerase Chain ReactionABSTRACT
h may play a role in the pathogenesis of SLE.
ABSTRACT
Objective To investigate two single nucleotide polymorphism sites of IRF5 and to de-tect their relationship with SLE in a population from Shandong province. Methods The polymorphisms (rs2004640 G/T,rs10954213 G/A) were detected with PCR-RFLP in 92 eases of SLE and 88 healthy con-trols. The genotype and allele frequencies were calculated and analyzed. Results The genotype frequencies Of GG, GT and TT in rs2004640 site in SLE were 0. 198, 0.521 and 0.281, respectively. The difference was significant between SLE and centrol (X2 = 8.73, P < 0.05). The genotype frequencies of GG, GA and AA in rs10954213 site in SLE were 0. 318, 0. 409 and 0.273, respectively. The differenee was significant between SLE and control (X2 = 6. 36, P < 0. 05). Conclusion The polymorphism of rs2004640, rs10954213 in IRF5 may be associated with SLE in the population of Han nationality from Shandong province of China.