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.
Base Sequence ; Cloning, Molecular ; Genetic Vectors ; genetics ; Mutagenesis, Site-Directed ; methods ; Nucleic Acid Amplification Techniques ; Plasmids ; Polymerase Chain Reaction

Objective: This project aimed to explore the effectiveness of estimating individual treatment effect on real data, among the heterogeneous population, with Causal Forests (CF) method, to find out the characteristics of heterogeneous population. Methods: We designed and conducted four computer simulation schemes to verify the effect of estimating on individual treatment, using the CF under four different environments of the treatment effects. Real data was then analyzed for the catheterization on right heart. Results: Results from the simulation process showed that the values on individual treatment effect that were estimated by causal forests were consistent with the population effect as well as in line with the expected distribution under the setting of four different effect values. Results of real data analysis showed that values of individual treatment effect among most patients appeared positive, so the use of RHC could cause an increase of the '180-day mortality rate’ in the sampled population. Patients with lower predicted probability of 2-mo survival and albumin were more likely to have a lower risk of death after using the RHC. Conclusion CF method could be effectively used to estimate the individual treatment effect and helping the individuals to make decision on the receipt of treatment.