基因表达差异显示实验

Double-strand breaks (DSBs) disrupt DNA integrity and cause genomic instability and cancer, mutations, or cell death. Among the pathways utilized by cells of higher eukaryotes to repair this lesion, nonhomologous end-joining (NHEJ) is the most dominant. The biochemical characteri ...

Gene targeting by homologous recombination in mammalian cells is an important tool for generating genetically modified mice used for modeling human diseases. Gene targeting approaches are also useful for studying the mechanisms of homologous recombination. We have developed ge ...

In this chapter, we describe a gene-specific quantitative polymerase chain reaction (QPCR)-based assay for the measurement of DNA damage, using amplification of long DNA targets. This assay has been extensively used to measure the integrity of both nuclear and mitochondrial genomes ex ...

Because of a substantial rise in the incidence of skin cancer in the United Kingdom and elsewhere a greater awareness of the role of sun-induced cutaneous genetic damage has developed. This, in turn, has increased interest in the cellular mechanisms responsible for tumorigenesis, and the need ...

There is a need to analyze the formation of DNA lesions in specific sequence contexts. The formation and repair of DNA damage at specific locations in the genome is modulated by the DNA sequence, by DNA methylation patterns, by the transcriptional status of the locus, and by chromatin proteins assoc ...

The method described here makes use of a polyclonal antiserum to measure repair of the principal photoproducts induced in DNA by short-wave ultraviolet light (UV-C)—pyrimidine-pyrimidone 6-4 photoproducts (PPs) and cyclobutane pyrimidine dimers (CPDs). DNA extracted from irra ...

The Fast Micromethod is a convenient and quick fluorimetric microplate assay for the assessment of DNA single-strand breaks and their repair. This method measures the rate of unwinding of cellular DNA on exposure to alkaline conditions using a fluorescent dye which preferentially binds ...

The electrophoretic mobility shift assay (EMSA) can be used to identify proteins that bind specifically to damaged DNA. EMSAs detect the presence of key DNA repair proteins, such as ultraviolet (UV)-damaged DNA binding protein, which is involved in nucleotide excision repair, and Ku and DNA- ...

We describe simple and efficient construction of mismatch repair (MMR) substrates, by generation of gapped plasmids using one sequence-specific nicking endonuclease (N.BstNBI), ligation of synthetic oligomers into the gaps, and introduction of defined single nicks for initia ...

Abasic sites in DNA arise under a variety of circumstances, including destabilization of bases through oxidative stress, as an intermediate in base excision repair, and through spontaneous loss. Their persistence can yield a blockade to RNA transcription and DNA synthesis and can be a sour ...

A rapid, convenient and safe in vitro assay system for base excision repair is described. Whole cell extracts are prepared by detergent-based cell lysis and provide a vigorous activity of AP site repair. A circular DNA substrate is used for detection of both DNA polymerase β-dependent and prolif ...

Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5′-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombinati ...

Methods are described for measuring nucleotide excision repair (NER) of damaged plasmid DNA using fractionated mammalian cell extracts. NER creates a single-stranded gap of approx 25–30 nt. Filling of this gap by repair synthesis can be monitored by the incorporation of radioactive nuc ...

Genetic alterations affecting nucleotide excision repair, the most versatile DNA-repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xerode ...

Analysis of the mechanism of nucleotide excision repair (NER) using cell-free extract systems and purified proteins requires DNA substrates containing chemically defined lesions that are placed at a unique site in a DNA duplex. In this way, NER can be readily and specifically measured by det ...

Proliferating cell nuclear antigen (PCNA) is a homotrimeric protein adopting a ring structure that may encircle DNA. In this form, PCNA functions as a sliding platform to which different types of catalytic and regulatory proteins are tethered to perform DNA transactions such as replicat ...

Significant advances have been made in identifying a complex network of proteins that could play a role in the repair of DNA damage in the context of chromatin. Insights into this process have been obtained by combining damaged DNA substrates with mammalian cell-free systems that contain both D ...

Different challenge assays have been used to investigate cellular responses following exposure to DNA damaging agents. Our protocol uses X- or γ-rays or ultraviolet light to challenge cells to repair the induced damage, and chromosome aberrations as a biomarker to indicate DNA repair pro ...

A number of ongoing delayed effects have now been described in the progeny of an irradiated cell. These are grouped under the rubric of radiation induced genomic instability. Perhaps the best characterized is the dynamic production of chromosomal rearrangements in some clonally expand ...

Extensive fragmentation of nuclear DNA occurs during apoptosis, and the presence of DNA strand breaks is considered to be a marker of the apoptotic mode of cell death. This chapter describes methods to label in situ DNA strand breaks with fluorochromes for detection by flow or laser scanning cyto ...