Heteroduplex cleavage assay for screening of probable zygosities resulting from CRISPR mutations in diploid single cell lines.

The most common gene editing methods, such as CRISPR, involve random repair of an induced double-stranded DNA break through the non-homologous end joining (NHEJ) repair pathway, resulting in small insertions/deletions. In diploid cells, these mutations can take on one of three zygosities: monoallelic, diallelic heterozygous, or diallelic homozygous. While many advances have been made in CRISPR delivery systems and gene editing efficiency, little work has been done to streamline detection of gene editing events.

Validation and long-term assessment of an approach for the high throughput determination of lipophilicity (log POW) values using multiplexed, absorbance-based capillary electrophoresis.

A critical evaluation of the use of 96-capillary multiplexed microemulsion electrokinetic chromatography (MMEEKC) for the indirect determination of octanol-water partition coefficients (log POW values) for a wide range of structurally different compounds is presented. The various components of the microemulsion solution were evaluated and optimized for use in a multiplexed capillary format. A six-component calibration mixture and 23 different solutes (n = 4 each) were analyzed simultaneously, providing a throughput of up to 46 samples/h, which translates to greater than a 20-fold improvement over existing indirect log POW methods.

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm.

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar.

Functional interactions between heterologously expressed starch-branching enzymes of maize and the glycogen synthases of Brewer's yeast.

Starch-branching enzymes (SBEs) catalyze the formation of alpha(1-->6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis.