基因表达差异显示实验

Despite increasing sequencing capacity, genetic disease investigation still frequently results in the identification of loci containing multiple candidate disease genes that need to be tested for involvement in the disease. This process can be expedited by prioritizing the can ...

Prioritization of most likely etiological genes entails predicting and defining a set of characteristics that are most likely to fit the underlying disease gene and scoring candidates according to their fit to this “perfect disease gene” profile. This requires a full understanding of the ...

In gene prediction, studying phenotypes is highly valuable for reducing the number of locus candidates in association studies and to aid disease gene candidate prioritization. This is due to the intrinsic nature of phenotypes to visibly reflect genetic activity, making them potentia ...

The discovery of RNAi, in which double-stranded RNA (dsRNA) suppresses the translation of homologous mRNA, has had a huge impact on evolutionary genetics by enabling the analysis of loss-of-function phenotypes in organisms in which classical genetic analysis is laborious or impossib ...

Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is emplo ...

Successful meiotic recombination is driven by a series of programmed chromosome dynamics that include changes in the protein composition of meiotic chromosomes and the juxtaposition of homologous chromosomes. The simultaneous visualization of both chromosome-bound prot ...

Recombination in first meiotic prophase is initiated by endogenous breaks in double-stranded DNA (DSBs) which occurs during a time when chromosomes are remodeled and proteinaceous cores (axes) are assembled along their length. DSBs are instrumental in homologue recognition and un ...

Homologous recombination is an important pathway for error-free repair of DNA lesions, such as single- and double-strand breaks, and for rescue of collapsed replication forks. Here, we describe protocols for live cell imaging of single-lesion recombination events in the yeast Sacchar ...

Nuclear organization is involved in numerous aspects of cellular function. In yeast, analysis of the nuclear position and dynamics of the silent and active mating-type loci has allowed to gain insight into the mechanisms involved in directing mating-type switching. The fluorescent re ...

Meiosis is initiated by the programmed formation of DNA double-strand breaks (DSBs). These DSBs are repaired by homologous recombination to promote crossover formation that ensures proper chromosomal segregation in meiosis. hRad51 and hDmc1 are two human recombinases present du ...

During homologous recombination and homology-directed repair of broken chromosomes, proteins that mediate and oppose recombination form dynamic complexes on damaged DNA. Quantitative analysis of these nucleoprotein assemblies requires a robust signal, which reports on t ...

Single-molecule studies of protein–DNA interactions continue to yield new information on numerous DNA processing pathways. For example, optical microscopy-based techniques permit the real-time observation of proteins that interact with DNA substrates, which in turn allows ...

A crucial process to ensure cell survival and genome stability is the correct replication of the genome. DNA replication relies on complex machinery whose mechanisms are being elucidated using different model systems. A major aspect of this process, which is an intense subject of investig ...

Rad51-mediated pairing between homologous DNA sequences during homologous recombination (HR) plays pivotal roles in DNA double-strand break repair. The multi-step process occurs through cooperation of Rad51 and a number of accessory protein factors. The development of various ...

Double-stranded DNA breaks (DSB), the most harmful type of DNA lesions, cause cell death and genome instability. Homologous recombination repairs DSB using homologous DNA sequences as templates. Here we describe a set of reactions that lead to reconstitution of the double-stranded DNA b ...

DNA strand exchange is a core reaction of homologous recombination directly catalyzed by Rad51/Dmc1 RecA family recombinases in eukaryotes. This reaction proceeds through the formation of several DNA intermediates. The X-shaped four-way DNA structure known as a Holliday junction ...

Homologous recombination (HR) is a high-fidelity DNA repair pathway that maintains genome integrity, by repairing double strand breaks (DSBs) and single-stranded DNA (ssDNA) gaps and by supporting stalled/collapsed replication forks. The RecA/Rad51 family of proteins are the key ...

Structure-selective nucleases perform DNA strand incisions crucial to the repair/resolution of branched DNA molecules arising during DNA replication, recombination, and repair. From a combination of genetics and in vitro nuclease assay studies, we are just beginning to underst ...

Biochemical reconstitution using purified proteins and defined DNA substrates is a key approach to develop a mechanistic understanding of homologous recombination. The introduction of sophisticated purification tags has greatly simplified the difficult task of purifyi ...

The immunoglobulin (Ig) genes of B cells are diversified at high rate by point mutations whereas the non-Ig genes of B cells accumulate no or significantly fewer mutations. Ig hypermutations are critical for the affinity maturation of antibodies for most of jawed vertebrates and also contri ...