遗传学实验技术

ϕC31 integrase is a site-specific recombinase from a bacteriophage that has become a useful tool in mammalian cells. The enzyme normally performs precise, unidirectional recombination between two attachment or att sites called attB and attP. We have shown that an attP site preintegrated ...

Recombinant polymerase chain reaction (PCR) (1) is the method of choice if one wants to modify a cloned DNA. It is a versatile technique that allows operations as different as creation of deletions, addition of small insertions, site-directed mutagenesis, and construction of chimeric mole ...

Conventional cDNA library construction often requires a minimum available amount of material (typically 1 or 2 μg of polyA+ RNA). For complex organs, such as brain, or certain species, such as humans, as well as subsets of cell types, this condition is often difficult to fulfill. Amplification by po ...

As more and more genes are cloned and sequenced, it is apparent that nearly all genes are related to other genes. Similar genes are grouped into families, such as the collagen and globin gene families. There are also gene superfamilies. Gene superfamilies are composed of genes that have areas of high ho ...

The polymerase chain reaction (PCR) (1) provides a rapid means for the recombination and site-directed mutagenesis of DNA (2). DNA modification can occur during PCR because the primers are incorporated into the ends of the PCR product. The simplest PCR-based method for site-directed mutage ...

RNA amplification is a series of molecular manipulations designed to amplify genetic signals from small quantities of starting materials (including single cells and homogeneous populations of individual cell types) for microarray analysis and other downstream genetic meth ...

Formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable resource for gene expression analysis, as they are the most widely available materials for studies of human disease. However, degradation of RNA during tissue fixation and storage makes FFPE-derived RNAs h ...

Proteins are the main actors in all physiological and pathological processes. Since the final structure of the protein does not depend on the DNA sequence or even the mRNA sequence alone, the search for direct approaches on the proteome has gained great interest. The most complex and probably the l ...

MicroRNAs (miRNAs) represent a class of small, noncoding RNAs. These small RNAs are involved in diverse biological processes, and it has been predicted that about one third of human messenger RNAs (mRNAs) appear to be miRNA targets, underlying the major influence of miRNAs on almost all cellular ...

Recent advances in antibody microarray technology have facilitated the development of multiplexed diagnostic platforms. Highly parallel antigen expression data obtained from these arrays allow disease states to be characterized using protein patterns rather than indiv ...

The recent introduction of two-dimensional fluorescent difference gel electrophoresis has enabled the large screening of differential protein expression levels with higher confidence and greater sensitivity than using the classical two-dimensional electrophores ...

A key issue in proteomics is to quantify changes in protein levels in complex biological samples under different conditions. Traditional two-dimensional gel (2-DE) electrophoresis-based proteomic approaches are tedious and suffer from several limitations, including diffi ...

“Gel-free,” or mass spectrometry (MS)-based, proteomics techniques are emerging as the methods of choice for quantitatively comparing proteins levels among biological proteomes, since they are more sensitive and reproducible than two dimensional gel (2-DE)-based methods. Cur ...

Two dimensional nano-high-performance liquid chromatography (nanoHPLC) coupled directly to a high-resolution tandem mass spectrometer (2D-nLC-MS/MS) is an excellent method for analyzing very complex peptide mixtures, especially when the quantity of sample available for ...

The increasing demand for production and characterization of diverse groups of recombinant proteins necessitates the analysis of several constructs and fusion tags in a variety of expression systems. The challenge is to screen multiple clones quickly for the desired properties. Wh ...

In many large genetic studies, the amount of available DNA can be one of the criteria for selecting samples for study. In the case of large population cohorts, selecting samples based on their DNA yield can lead to biased sample selection. In addition, many valuable clinical and research sample coll ...

The mammalian two-hybrid system is a very powerful tool to investigate protein-protein interactions in terms of functional domains and identify potential binding ligands and partners of a protein. Compared with the yeast two-hybrid system, the mammalian two-hybrid system provides ...

Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry—based prot ...

Defining protein-protein interaction networks is a major goal of proteomics. Here, we present a protocol for coimmunoprecipitation, a technique suitable for the isolation of whole protein complexes in vivo and their subsequent identification by either immunoblotting or mass spe ...

Protein fragment complementation has emerged as a powerful tool for measuring protein-protein interactions in the context of live cells. The adaptation of this strategy for use with firefly luciferase now allows for the non-invasive, quantitative, real-time readout of protein inte ...