分子生物学

Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, the inheritance of specific methylation patterns can be studied. Here, we present a protocol for analyzing DNA methylation patterns through MSAP markers in potato interspecific hybrids and their parental genotypes.

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

Capillary electrophoresis (CE) is identified as a promising technology for the study of nucleic acid molecules because of its high efficiency, high throughput with automation and integration. Compared to the traditional method of slab gel electrophoresis (SGE), the advantages of CE cannot be emphasized more. Most of CE process, including sample injection, detection and data analysis, is able to be automated which will save great labor for industrial and research labs. CE used the separation channel with micrometer-scale diameter, so the joule heat is easy to be dissipated during electrophoresis. Thus high separation voltage (> 100 V/cm) is allowed in CE while in SGE (usually ~10 V/cm) it usually causes severe band broadening. Because the band broadening is restrained efficiently in CE, it is capable of detecting minute samples and becoming more sensitive than SGE. The advantage of allowing high voltage consequently speeds up the CE separation and yields a better throughput compared to SGE. CE costs less reagents, for example buffer solutions, sieving matrix, dye reagents etc. In addition, the micrometer-scale channel is easy to be integrated with upstream and downstream sample treatment units, forming a lab on a chip. This merit of CE already attracted considerable interests among researchers from various areas.

The difficulties of CE involve filling the gels (agarose or cross-linked polyacrylamide) into the capillary tube. Also, the reproducibility and the life-time of the gel-capillary are limited. But the small-diameter capillary allows to use replaceable polymer solutions, which can efficiently prevent the convection of the separation buffer. Polymer solutions are easier to be filled into the capillary and yield more stable separations. Thus, those difficulties are resolved by doing capillary polymer electrophoresis (CPE), which is going to be described in this protocol.

Several separation modes, for example, capillary gel electrophoresis (CGE), CPE, capillary zone electrophoresis (CZE), capillary isotachophoresis (CITP) and so on, have been developed for analysis of different kinds of molecules. Here, we introduce the protocol for CPE in detail, which is for the separation of dsDNA, dsRNA (including siRNA) molecules. Polymer solutions are filled into the capillary as a sieving matrix for double strand nucleic acids separation. Hydroxyethylcellulose (HEC) polymer is employed as the sieving polymer in this case. A home-built CE system is described in detail.

DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA laddering. We herein describe a protocol to detect a programmed cell death-associated DNA laddering of plant tissues. After agarose-gel electrophoresis of genomic DNA, Southern hybridization using DIG-labeled genomic DNA probe is performed, that improves detection of DNA laddering.

Mice are extremely powerful mammalian genetic model organisms for basic and medical research, but managing a colony of transgenic mice is time consuming and expensive, many times requiring the help of dedicated technicians. Slow and laborious genotyping procedures add to the hassle. Outsourcing is costly and may not be as fast as desired, especially when setting up time sensitive experiments. Ultrafast genotyping protocols often require real-time PCR instruments and commercial reagents that may not be economical or practical. This protocol, adapted from methods suggested by The Jackson Laboratory, employs a minimalist approach that maximizes convenience by simplifying the tissue digestion/DNA extraction process and using a high-speed electrophoresis system for sample analysis. Genotype PCR results can be obtained in 3 h or less for as many samples as can fit in a PCR machine or can be efficiently handled by a user. Subsequent ethanol or chloroform purified DNA can be used in a standard PCR reaction to roughly identify a homozygous and a hemizygous mouse.

Two-dimensional agarose gel electrophoresis (2D gel) analysis is used extensively as a method to detect origins of replication. Here, I present a simplified method for the isolation of yeast genomic DNA for 2D gel analysis from a small number of yeast cells. This DNA isolation method is simpler and less time consuming than the traditional method that involves CsCl density gradient centrifugation. This method could be modified for 2D gel analysis in other organisms as well.