遗传学实验技术

The in vitro DNA mismatch repair (MMR) assay is a very useful technique for studying the functions and the mechanisms of the MMR system in genome maintenance. This assay has been effectively used to evaluate MMR proficiency in various tumor cells and to identify the majority of the protein componen ...

Mismatch repair corrects biosynthetic errors generated during DNA replication. Mismatch repair deficiency causes a mutator phenotype and directly underlies hereditary nonpolyposis colorectal cancer and some sporadic cancers. Because of remarkably high conservation ...

Solar ultraviolet radiation (UVR), through the formation of DNA photolesions, is the primary cause of most skin cancers. A better understanding of the mechanisms of UVR-induced DNA damage may help prevent skin cancer and this may be achieved using methods to quantify DNA damage. The immuno-sl ...

Over the past 25 years, the use of polyclonal and monoclonal antibodies to quantify DNA damage has burgeoned. Immunoassays offer distinct advantages over other analytical procedures currently used to measure DNA damage including adaptability, sensitivity, and selectivity. This ...

The formation and repair of DNA damage at specific locations in the genome is modulated by DNA sequence context, by DNA cytosine-5 methylation patterns, by the transcriptional status of the locus and by proteins associated with the DNA. The only method currently available to allow precise sequ ...

Interstrand cross-links (ICL) are one of the most hazardous types of DNA damage as they form a roadblock to all processes that involve strand separation. Repair of these lesions involves several different DNA repair pathways, but the molecular mechanism is unclear. Here we describe a system th ...

Plasmids containing a site-specific DNA interstrand cross-link (ICL) are invaluable tools for the investigation of ICL repair pathways at the biochemical and cellular level. We describe a procedure for preparation of plasmid DNA substrates containing a single ICL at a specific site. The ...

Base excision repair (BER) is a major pathway for the removal of endogenous and exogenous DNA damage. This repair mechanism is initiated by DNA glycosylases that excise the altered base, and continues through alternative routes that culminate in DNA resynthesis and ligation. In contrast to t ...

Base excision repair (BER) is an essential cellular mechanism that maintains genome stability by repairing DNA damage, such as DNA base lesions, base loss (AP sites) and single strand breaks, generated through endogenous metabolism or via exogenous mutagens. Therefore, in vitro BER assa ...

Base excision repair (BER) is an important mechanism to maintain genomic stability. Here we offer a set of protocols to quantitatively analyze BER capacity in whole cell-free yeast extracts. Cell-free yeast extracts were obtained by a French press procedure and repair capacities were mea ...

Chromosomal double strand breaks (DSBs) can be repaired by a number of mechanisms that result in diverse genetic outcomes. To examine distinct outcomes of chromosomal DSB repair, a panel of human cell lines has been developed that contain GFP-based reporters with recognition sites for the ra ...

The chicken B lymphocyte cell line DT40 is now a well-established system for studying DNA repair in vertebrates. Central to its utility is its ready genetic tractability allowing for the creation of isogenic mutants in any chosen gene. The combination of this genetic tractability with the I-Sc ...

DNA double-strand breaks (DSBs) have proven to be very potent initiators of recombination in yeast and other organisms. A single, site-specific DSB initiates homologous DNA repair events such as gene conversion, break-induced replication, and single-strand annealing, as well as nonh ...

Single-stranded DNA (ssDNA) is a DNA repair, replication, and recombination intermediate and a stimulus for checkpoint kinase-dependent cell cycle arrest. Current assays to detect ssDNA generated in vivo are indirect, laborious, and generally require the use of radioactivity. Here, ...

Single-stranded DNA (ssDNA) intermediates play an important role in processes such as DNA replication and homologous recombination, DNA damage responses, and DNA repair. Using quantitative amplification of ssDNA (QAOS), ssDNA arising during various cellular processes in comp ...

Pulsed-field gel electrophoresis (PFGE) is a technique for resolving large (up to 10 Mb) DNA molecules. Using multiple pairs of electrodes DNA is subject to an alternating electric field through a solid agarose matrix. As the current changes direction the reorientation time of DNA is proport ...

The ability to detect and measure DNA single-strand breaks has been the aim of numerous assays developed to assess genotoxicity. These methods often rely on alkaline conditions to denature DNA. However, alkaline treatment of DNA also introduces artifactual SSBs through the cleavage of al ...

The Flp-nick system is a simple in vivo system developed for studying the cellular responses to a protein-bound nick at a single genomic site in the budding yeast Saccharomyces cerevisiae. The Flp-nick system takes advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp rec ...

Mitochondrial DNA (mtDNA) is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to be partic ...

Topoisomerase II is an essential enzyme that is required for a number of critical nuclear processes. All of the catalytic functions of topoisomerase II require the enzyme to generate a transient double-stranded break in the backbone of the double helix. To maintain genomic integrity during ...