遗传学实验技术

Denaturing gradient gel electrophoresis (DGGE) is a powerful mutation detection technique described by Fisher et al. in 1983 (1). It allows the resolution of relatively large DNA fragments (usually polymerase chain reaction products) differing by only a single nucleotide. DGGE is most f ...

Defects of the factor VIII gene causes (f8) hemophilia A, an hemorrhagic X-linked disorder. The factor VIII gene is 186 kb long with 26 exons, varying from 69 bp (exon 5) to 3106 bp (exon 14) (1). The factor VIII mRNA is 9028 bases in length with a coding region of 7053 nucleotides (2).

Single-stranded DNA will recognize a complementary strand with high specificity under suitably controlled conditions. In situ hybridization (ISH) exploits this phenomenon by hybridizing an appropriately labeled singlestranded DNA “probe” to target sequences in situ in eit ...

The development of the polymerase chain reaction (PCR) has allowed the rapid isolation of DNA sequences utilizing the hybridization of two oligonucleotide primers and subsequent amplification of the intervening sequences by Taq polymerase. This technique has facilitated the id ...

Analysis of DNA fragmentation using Terminal deoxynucleotidyl Transferase (TdT)-mediated nick end-labeling (TUNEL) is a very sensitive technique for in situ detection of various types of DNA breaks in cells undergoing apoptosis and/ or necrosis (1–6). TUNEL technique is widely used, ...

Chromatin structure can affect the level of induced DNA damage and/or repair, and so these effects may vary within different DNA sequence areas of a cell, as well as between different cell types. DBD-FISH is a procedure that allows both intragenomic and intercellular heterogeneity in DNA break ...

Stroke is a major cause of disability in the United States affecting approximately 500,000 people per year at a rate of one person every minute. Stroke induces a decrease in ATP, an increase in the expression of immediate early genes, and causes oxidative damage to DNA, RNA, protein and lipid. One determ ...

The heat-stable DNA polymerase utilized in the polymerase chain reaction (PCR) has been widely used to amplify DNA fragments since its conception by Kerry Mullis in 1985. However, it soon became apparent that there was a constraint on the maximum size of amplified fragments. For genomic DNA, this w ...

Many single-base substitutions that lead to inherited diseases, the predisposition to genetic disorders, and cancer are increasingly being discovered. The ability to amplify specific DNA sequences by the polymerase chain reaction (PCR) (1) has made it possible to rapidly and accurat ...

In 1983, the Cetus scientist Kary Mullis developed an ingenious “in vitro” nucleic acid amplification technique termed the polymerase chain reaction (PCR). This technique involves the use of a pair of short (usually 20 bp long) pieces of synthesized DNA called primers and a thermostable DNA po ...

Phosphorylation is one of the most abundant post-translational modifications on protein and one that frequently has functional biological consequences. For this reason, screening protein samples for phosphorylations has become an important tool in biochemical research. Af ...

Producing high quality two-dimensional electrophoretic gels of bacterial cell membrane proteins is challenging because of non-membrane protein impurities within cell membrane preparations and the intractability of membrane proteins for solubilisation. Incubation ...

Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000–50,000 proteins are encoded by the human genome underlines the importance of posttranslational mod ...

Methods that combine efficient solubilization with enrichment of proteins and intact protein complexes are of central interest in current membrane proteomics. We have developed methods based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins ...

Because lipid rafts are plasma membrane platforms mediating various cellular events such as in signal transduction, immunological response, pathogen invasion, and neurodegenerative diseases, protein identification in the rafts could provide important information to st ...

Blue native PAGE is a discontinuous electrophoretic system that allows the separation of membrane protein complexes in a native, enzymatically active state with high resolution. Membrane protein complexes are solubilized by neutral, nondenaturing detergents like n-dodecyl- ...

Procurement of pure populations of cells from heterogeneous histological sections can be accomplished utilizing tissue microdissection. At present, a variety of different manual and laser-based dissection tools are available and each method has particular strengths and wea ...

Hydrophilic interaction chromatography (HILIC) is a variant of normal phase chromatography, in which analyte molecules attach to a solid support (e.g., poly aspartamide silica) by the action of a mobile phase containing a high amount of organic modifier such as acetonitrile or propanol. E ...

Stable isotope labeling in combination with mass spectrometry has emerged as a powerful tool to identify and relatively quantify thousands of proteins within complex protein mixtures. Here we describe a method, termed isotope-coded protein label (ICPL), which is capable of high-thro ...

Prefractionation procedures not only aid in reducing sample complexity, but also permit loading of higher protein amounts within the separation range applied to two-dimensional electrophoresis (2-DE) gels and thus facilitate the detection of less abundant protein species. Hen ...