We describe a simple method of using rolling circle amplification to amplify vector DNA such as M13 or plasmid DNA from single colonies or plaques. Using random primers and 29 DNA polymerase, circular DNA templates can be amplified 10,000-fold in a few hours. This procedure removes the need for lengthy growth periods and traditional DNA isolation methods. Reaction products can be used directly for DNA sequencing after phosphatase treatment to inactivate unincorporated nucleotides. Amplified products can also be used for in vitro cloning, library construction, and other molecular biology applications.A fundamental requirement for molecular biology is the isolation and amplification of specific DNA sequences. Target sequences are typically inserted into circular vectors, propagated in a biological host, and isolated by physical methods (Sambrook et al. 1989). However, such methods are laborious, costly, and not amenable to high-density formats. PCR is also used to amplify defined sequences, but can introduce sequence errors and is limited to amplification of short DNA segments (Innis et al. 1990).In nature, the replication of circular DNA molecules such as plasmids or viruses frequently occurs via a rolling circle mechanism (Kornberg and Baker 1992). As a laboratory method, linear rolling circle amplification (RCA) (Fire and Xu 1995;Liu et al. 1996;Lizardi et al. 1998) is the prolonged extension of an oligonucleotide primer annealed to a circular template DNA. A continuous sequence of tandem copies of the circle is synthesized. RCA has the advantage of not requiring a thermal cycling instrument. Two primers are used to perform exponential (or hyperbranched) RCA, one for each strand (Lizardi et al. 1998). A cascade of strand displacement reactions results in an exponential amplification.Previously, RCA had been used to amplify small DNA circles approximately 100 nt in length. However, the rate for plasmid-sized targets is only about 20 copies per hour, limiting the usefulness with plasmids or other circles larger than 0.2 kb. We describe here a technique called multiply-primed rolling circle amplification (multiply-primed RCA) that uses the unique properties of 29 DNA polymerase and random primers to achieve a 10,000-fold amplification. This robust process allows amplification of circular DNA directly from cells or plaques, generating high-quality template for use in DNA sequencing, probe generation, or cloning. The method is simple and is optimally performed at 30°C, making it suitable for a variety of applications. This will make it attractive for high-throughput processes and 384-well formats. RESULTS Increased Yield in RCA Using Random Hexamer PrimersIn multiply-primed RCA, the use of multiple primers annealed to a circular template DNA generates multiple replication forks (Fig. 1). RCA proceeds by displacing the nontemplate strand. In this way, product strands are "rolled off" of the template as tandem copies of the circle. Random priming allows synthesis of both strands, resulting in double-stranded product. ...

The REV3 and REV7 genes of the yeast Saccharomyces cerevisiae are required for DNA damage-induced mutagenesis. The Rev3 and Rev7 proteins were shown to form a complex with DNA polymerase activity. This polymerase replicated past a thymine-thymine cis-syn cyclobutane dimer, a lesion that normally severely inhibits replication, with an efficiency of approximately 10 percent. In contrast, bypass replication efficiency with yeast DNA polymerase alpha was no more than 1 percent. The Rev3-Rev7 complex is the sixth eukaryotic DNA polymerase to be described, and is therefore called DNA polymerase zeta.

Mutagenesis induced by DNA damage in Saccharomyces cerevisiae requires the products of the REV1, REV3 and REV7 genes. The Rev3 and Rev7 proteins are subunits of DNA polymerase-zeta (Pol-zeta), an enzyme whose sole function appears to be translesion synthesis. Rev1 protein has weak homology with UmuC protein which facilitates translesion synthesis in Escherichia coli by an unknown mechanism. We show here that Rev1 protein has a deoxycytidyl transferase activity which transfers a dCMP residue from dCTP to the 3' end of a DNA primer in a template-dependent reaction. Efficient transfer occurred opposite a template abasic site, but approximately 20% transfer also occurred opposite a template guanine and approximately 10% opposite adenine or uracil; < or = 1% was seen opposite thymine or cytosine. Insertion of cytosine opposite an abasic site produced a terminus that was extended efficiently by Pol-zeta, but not by yeast Pol-alpha.

SummaryThe function of the Saccharomyces cerevisiae REV1 gene is required for translesion replication and mutagenesis induced by a wide variety of DNAdamaging agents. We showed previously that Rev1p possesses a deoxycytidyl transferase activity, which incorporates dCMP opposite abasic sites in the DNA template, and that dCMP insertion is the major event during bypass of an abasic site in vivo. However, we now find that Rev1p function is needed for the bypass of a T±T (6±4) UV photoproduct, a process in which dCMP incorporation occurs only very rarely, indicating that Rev1p possesses a second function. In addition, we find that Rev1p function is, as expected, required for bypass of an abasic site. However, replication past this lesion was also much reduced in the G-193R rev1-1 mutant, which we find retains substantial levels of deoxycytidyl transferase activity. This mutant is, therefore, presumably deficient principally in the second, at present poorly defined, function. The bypass of an abasic site and T±T (6±4) lesion also depended on REV3 function, but neither it nor REV1 was required for replication past the T±T dimer; bypass of this lesion presumably depends on another enzyme.

Aims Despite the effects of statins in reducing cardiovascular events and slowing progression of coronary atherosclerosis, significant cardiovascular (CV) risk remains. Icosapent ethyl (IPE), a highly purified eicosapentaenoic acid ethyl ester, added to a statin was shown to reduce initial CV events by 25% and total CV events by 32% in the REDUCE-IT trial, with the mechanisms of benefit not yet fully explained. The EVAPORATE trial sought to determine whether IPE 4 g/day, as an adjunct to diet and statin therapy, would result in a greater change from baseline in plaque volume, measured by serial multidetector computed tomography (MDCT), than placebo in statin-treated patients. Methods and results A total of 80 patients were enrolled in this randomized, double-blind, placebo-controlled trial. Patients had to have coronary atherosclerosis as documented by MDCT (one or more angiographic stenoses with ≥20% narrowing), be on statin therapy, and have persistently elevated triglyceride (TG) levels. Patients underwent an interim scan at 9 months and a final scan at 18 months with coronary computed tomographic angiography. The pre-specified primary endpoint was changed in low-attenuation plaque (LAP) volume at 18 months between IPE and placebo groups. Baseline demographics, vitals, and laboratory results were not significantly different between the IPE and placebo groups; the median TG level was 259.1 ± 78.1 mg/dL. There was a significant reduction in the primary endpoint as IPE reduced LAP plaque volume by 17%, while in the placebo group LAP plaque volume more than doubled (+109%) (P = 0.0061). There were significant differences in rates of progression between IPE and placebo at study end involving other plaque volumes including fibrous, and fibrofatty (FF) plaque volumes which regressed in the IPE group and progressed in the placebo group (P &lt; 0.01 for all). When further adjusted for age, sex, diabetes status, hypertension, and baseline TG, plaque volume changes between groups remained significantly different, P &lt; 0.01. Only dense calcium did not show a significant difference between groups in multivariable modelling (P = 0.053). Conclusions Icosapent ethyl demonstrated significant regression of LAP volume on MDCT compared with placebo over 18 months. EVAPORATE provides important mechanistic data on plaque characteristics that may have relevance to the REDUCE-IT results and clinical use of IPE.