Previous studies of serial cloning in animals showed a decrease in efficiency over repeated iterations and a failure in all species after a few generations. This limitation led to the suggestion that repeated recloning might be inherently impossible because of the accumulation of lethal genetic or epigenetic abnormalities. However, we have now succeeded in carrying out repeated recloning in the...

Mammalian oocytes can reprogram somatic cells into a totipotent state enabling animal cloning through somatic cell nuclear transfer (SCNT). However, the majority of SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we identify histone H3 lysine 9 trimethylation (H3K9me3) of donor cell genome as a major barrier for efficient reprogramming by SCNT. Comparative tra...

Molecular cloning methods based on primer and overlap-extension PCR are widely used due to their simplicity, reliability, low cost and high efficiency. In this article, an efficient mega primer-mediated (MP) cloning strategy for chimaeragenesis and long DNA fragment insertion is presented. MP cloning is a seamless, restriction/ligation-independent method that requires only three steps: (i) the ...

Over the past decade, the focus of cloning has shifted from constructing plasmids that express a single gene of interest to creating multigenic constructs that contain entire pathways or even whole genomes. Traditional cloning methods that rely on restriction digestion and ligation are limited by the number and size of fragments that can efficiently be combined. Here, we focus on the use of hom...

USER cloning is a fast and versatile method for engineering of plasmid DNA. We have developed a user friendly Web server tool that automates the design of optimal PCR primers for several distinct USER cloning-based applications. Our Web server, named AMUSER (Automated DNA Modifications with USER cloning), facilitates DNA assembly and introduction of virtually any type of site-directed mutagenes...

Large deviations for additive path functionals of stochastic processes have attracted significant research interest, in particular the context particle systems and statistical physics. Efficient numerical `cloning' algorithms been developed to estimate scaled cumulant generating function, based on importance sampling via cloning rare event trajectories. So far, attempts study convergence proper...

The creation of genome-scale clone resources is a difficult and costly process, making it essential to maximize the efficiency of each step of clone creation. In this review, we compare the available commercial and open-source recombinational cloning methods with regard to their use in creating comprehensive open reading frame (ORF) clone collections with an emphasis on the properties requisite...