High-throughput conformation-sensitive gel electrophoresis for discovery of SNPs

High-throughput screening for single nucleotide polymorphisms (SNPs) or mutations can be achieved by inexpensive technologies. We modified the original protocols of conformation-sensitive gel electrophoresis (CSGE) to increase throughput several fold to 1.3 samples/min, which is about five times faster than denaturing high-performance liquid chromatography (DHPLC). The modifications include decreasing the gel thickness, increasing the number of lanes to 96, and increasing the number of samples per lane to seven. This high-throughput CSGE method is fast, robust, and as simple as the original protocols. Together with a two-stage strategy for screening homozygotes and the replacement of ethidium bromide with SYBR Gold DNA dye staining, this protocol is a reliable and cost-effective alternative for laboratories that require high-throughput screening.     

Agarose gel electrophoresis of denatured RNA with silver staining

This paper describes agarose gel electrophoresis and silver staining of denatured RNAs. Glyoxal- or formaldehyde-denatured RNAs are electrophoresed in an agarose gel cast on a plastic support using an inert, low conductivity buffer. Following electrophoresis, the gel is stained with a sensitive silver stain. The method produces sharp, well-resolved bands and yields accurate RNA size estimates. Because of its sensitivity and simplicity, it is suitable for routine laboratory use.     

Improvements of DNA sequencing gels

We describe three simple modifications of DNA sequencing gels which all result in improved oligonucleotide resolution as visualized by autoradiography. First, it was possible to reduce the thickness of the gel to 0.2 mm by using new gel molding techniques. Second, the gel could be dried without any distortions of its dimensions by prior binding of the gel to the surface of the glass plate. Third, a uniform high temperature was obtained in all parts of the gel during electrophoresis by replacing one of the glass plates with an inexpensive thermostating plate with circulating water. The use of this heating plate resulted in a straight band pattern all over the gel and also in the resolution of such bands which were not resolved in other electrophoresis systems.     

Analysis of tubulin isoforms by two-dimensional gel electrophoresis using SDS-polyacrylamide gel electrophoresis in the first dimension.

A two-dimensional electrophoretic system using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the first dimension and isoelectric focusing (IEF) in the second dimension was devised. In spite of its simplicity, this method could show a markedly high resolution for tubulin isoforms and moreover could classify them into alpha- or beta-tubulin as a two-dimensional profile. With this method, seven alpha- and four beta-tubulin isoforms could be detected within axoneme from Tetrahymena cilia. Moreover this method could also resolve tubulin isoforms from the rabbit brain. These results indicate that the present two-dimensional gel electrophoresis is a useful tool for the electrophoretic analysis of tubulin isoforms from various sources.     

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.     

Detection of serum proteins by native polyacrylamide gel electrophoresis using Blue Sepharose CL-6B-containing stacking gels

Analysis of serum proteins by native polyacrylamide gel electrophoresis is difficult because albumin is abundant in serum and interferes with the resolution of other proteins, especially alpha-antitrypsin which has mobility that is very similar to that of albumin. We present here a method in which serum proteins are separated by polyacrylamide gel electrophoresis using stacking gels containing Blue Sepharose CL-6B, which has a high affinity for albumin, lipoproteins, kinases, and pyridine-nucleotide-dependent oxidoreductases. During electrophoresis, proteins that bind to Blue Sepharose CL-6B stay in the stacking gel and do not migrate into the separating gel. As a consequence, certain proteins, including alpha(1)-antitrypsin, can be detected as clear bands. This method overcomes the requirement for fractionation of serum samples prior to electrophoresis to remove albumin and allows the simultaneous analysis of many samples.     

Cloning of the human type XVII collagen gene (COL17A1), and detection of novel mutations in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal variant of junctional epidermolysis bullosa (JEB). Previous findings have suggested that type XVII collagen is the candidate gene for mutations in this disease. We now have cloned the entire human type XVII collagen gene (COL17A1) and have elucidated its intron-exon organization. The gene comprises 56 distinct exons, which span approximately 52 kb of the genome, on the long arm of chromosome 10. It encodes a polypeptide, the alpha1(XVII) chain, consisting of an intracellular globular domain, a transmembrane segment, and an extracellular domain that contains 15 separate collagenous subdomains, the largest consisting of 242 amino acids. We also have developed a strategy to identify mutations in COL17A1 by use of PCR amplification of genomic DNA, using primers placed on the flanking introns. The PCR products are scanned for sequence variants by heteroduplex analysis using conformation-sensitive gel electrophoresis and then are subjected to direct automated sequencing. We have identified several intragenic polymorphisms in COL17A1, as well as mutations, in both alleles, in two Finnish families with GABEB. The probands in both families showed negative immunofluorescence staining with an anti-type XVII collagen antibody. In one family, the proband was homozygous for a 5-bp deletion, 2944del5, which resulted in frameshift and a premature termination codon of translation. The proband in the other family was a compound heterozygote, with one allele containing the 2944del5 mutation and the other containing a nonsense mutation, Q1023X. These results expand the mutation database in different variants of JEB, and they attest to the functional importance of type XVII collagen as a transmembrane component of the hemidesmosomes at the dermal/epidermal junction.     

水稻种子贮藏谷蛋白的改良电泳分析法

利用15％－25％丙烯酰胺梯度凝胶的SDS－PAGE分析法可将水稻种子贮藏谷蛋白分离为3个酸性（α）亚基和3个碱性（β）亚基,通过调节两性电解质比例对现有等用点了和焦电泳分析法进行改良,可将谷蛋白酸性亚基和碱性亚基分别分划为13和14条多肽带,将上述两种方法结合起来的双向电泳分析法可以高清晰度地离析谷蛋白并获得单一多肽,此改良的电泳分析系统有助于确定水稻谷蛋白变异及谷蛋白的生化研究。     

High throughput fluorescence-based conformation-sensitive gel electrophoresis (F-CSGE) identifies six unique BRCA2 mutations and an overall low incidence of BRCA2 mutations in high-risk BRCA1-negative breast cancer families

Mutational analysis of cancer susceptibility genes has opened up a new era in clinical genetics. In this report we present the results of mutational analysis of the BRCA2 coding sequences in 105 high-risk individuals affected with breast cancer and/or ovarian cancer and previously found to be negative for mutations of the BRCA1 coding sequence in our laboratory. These individuals have a positive family history with three or more cases of breast cancer and/or ovarian cancer at any age from the same side of the family tree. In order to perform a high throughput and reliable mutational analysis of the BRCA genes, we have adapted the conformation-sensitive gel electrophoresis mutation-scanning assay to a fluorescent platform. The advantages are speed, reproducibility and enhanced resolving power of the scanning method. Four unique mutations, including one missense and three frameshift mutations, were identified in the pool of 60 non-Jewish patients (7%). Two cases of the 6174delT mutation were identified in the 45 Ashkenazi Jewish individuals studied (5%). In addition, two novel frameshift mutations, not characteristic of the Jewish subgroup, were identified. Thus there were four mutations in total in this ethnic subgroup (9%). The six mutations identified in this combined patient pool, excluding the 6174delT mutations, are novel and have not been previously reported in the Breast Cancer Information Core (BIC) database. The results indicate that BRCA2 mutations account for the disease in less than 10% of this patient population. In addition, there is no significant difference in frequency of BRCA2 mutations between the Ashkenazi Jewish and non-Jewish families in our clinical patient pool. Received: 8 December 1997 / Accepted: 16 February 1998     

Reduced histone levels induced by "reeler" and "weaver" mutations in the mouse cerebellum.

Five major protein bands present in the polyacrylamide gel electrophoresis pattern of normal cerebellum are apparently absent or decreased in amounts in both reeler and weaver mutant cerebellar tissues. All five bands were identified as histones and the deficiencies related to the decreased cerebellar cellularity produced by both mutations. These results, therefore, rule out an earlier suggestion that two of these proteins are granular cell specific proteins (1). Preliminary evidence for high levels of F1 histone in the nuclei of cerebellar cells, which appear to be reduced in the reeler syndrome, is presented.     

小麦幼穗蛋白质双向电泳条件的优化

本研究以温光敏小麦为试材,用TCA/丙酮和酚提取法提取小麦幼穗蛋白样品,进行了双向电泳优化分析,并对双向电泳过程中出现的问题进行了讨论。结果表明,用TCA/丙酮法提取小麦幼穗蛋白质其产率(浓度)高于酚提取法。SDS-PAGE电泳显示,用TCA/丙酮提取法提取的蛋白质能获得较清晰条带,分辨率较高,而酚提取法提取的蛋白质其条带模糊,分辨率低。对蛋白质纯化除盐可以提高分辨率,减少横竖纹,获得背景清晰的圆形蛋白点。通过ImageMasterTM 2D Platinum5.0软件分析凝胶图谱,结果显示纯化后可降低噪点,纯化后蛋白点数可从未纯化蛋白点数的216增加到583。显然,采用TCA/丙酮法可获得高浓度高质量的蛋白质,而进一步纯化、除盐离子可进一步获得背景清晰可高重复性的电泳图谱。在双向电泳实验过程中,观察到一些异常缺陷胶的出现,如双向电泳图谱中蛋白点扩散,蛋白聚集形成斑点串,没有点或点很少,出现纵纹横纹及图谱扭曲等影响图谱质量的严重问题,本研究对这些问题做了分析并提出了解决方案。     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

Adaptation of conformation-sensitive gel electrophoresis to an ALFexpress DNA sequencer to screen BRCA1 mutations

Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mutations with accuracy. This is important in genes such as BRCA1 and BRCA2, in which alterations span the entire gene. We have adapted the CSGE assay to a fluorescent platform--a DNA sequencer one-color technology--that reduces the time involved and enhances resolving power for the complete scanning of the BRCA genes. Electrophoresis has high sensitivity and is performed in less than three hours, and the gel does not require staining with ethidium bromide. Eighteen single-base and six frameshift mutations in the BRCA1 gene were analyzed. We compared the manual and fluorescent CSGE methods, and all mutations were detected with accuracy.     

Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy

The secondary structure of adenylate kinase (EC 2.7.4.3) from E. coli was investigated under various conditions using Fourier transform infrared spectroscopy. The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is alpha-helical. A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% alpha-helix, 26% beta-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures. Binding of nucleotide (e.g., ATP) to the donor site decreases the beta-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein. These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin. The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed.     

Comparative studies of pig platelet membrane proteins

The proteins and glycoproteins of pig platelet membranes have been studied using gel electrophoretic techniques. A nomenclature is suggested from the apparent molecular weights estimated by one-dimensional electrophoresis. Isoelectric focusing showed that the majority of the proteins are in the 4.0-7.0 pH range. Subunits have been inferred from oligoproteins by two-dimensional, reduced-nonreduced, electrophoresis techniques. High resolution two dimensional electrophoresis combining isoelectric focusing and sodium dodecyl sulphate allows the observations of 60 polypeptide bands. An identification of some of those bands based on a correlation from reported human blood platelet membrane proteins is presented for comparison.     

Evidence for membrane-bound oligomerization of bacteriophage phi X174 lysis protein-E

The expression of cloned bacteriophage phi X174 lysis gene E was analyzed in minicells of Escherichia coli using two-dimensional gel electrophoresis. Beside the 10-11-kDa protein-E, at least two additional protein bands were detected, associated with the inner membrane, which showed the same isoelectric point as E. To clarify whether these proteins were E-specific, two different antibodies directed against a beta-galactosidase-E' hybrid protein and a synthetic oligopeptide corresponding to the C-terminal end of protein-E were raised. Immunoadsorption studies with anti-peptide-specific antibodies resulted in the detection of protein-E as well as in the detection of proteins of higher molecular weight. Two of these protein bands were positively recognized by anti beta-galactosidase-E' antibodies. The latter protein bands had the same molecular weight as the putative protein-E bands detected by two-dimensional gel electrophoresis indicating that these bands represent protein-E-specific oligomers. These data support the idea that an E-specific oligomeric structure penetrating the inner and outer membrane of E. coli is formed during the lytic action of protein-E.     

A high-pressure sample cell for circular dichroism studies.

The construction and operation of a preparative polyacrylamide-gel electrophoresis system is described. Emerging protein bands are collected by an intermittent pumping system which is based on the design of Brownstone ((1969) Anal. Biochem.27, 25–46). The original pressure-sensitive operation was, however, simplified to time-volume operation. Cooling of the gel by a central cooling finger, essentially according to Jovin et al. ((1964) Anal. Biochem.9, 351–369), has also been added. To accomodate the polyethylene tubing needed for intermittent collection of protein and also the central cooling finger, it is necessary to polymerize the gel in a mold before it is installed in the gel housing compartment of the electrophoresis cell. Gel concentrations of 5% and higher can be used in this system. Dilution of emerging protein samples by the intermittent collection system is kept to a minimum. This fact, together with simplicity of design makes it suitable for general preparative work with polyacrylamide-gel electrophoresis. Operation of the apparatus and resolution of protein bands are demonstrated by separation of bovine serum albumin polymers and thyroxine-binding proteins in human serum.     

An improved method for Southern DNA and Northern RNA blotting using a Mupid-2 Mini-Gel electrophoresis unit

An improved method for Southern DNA and Northern RNA blotting using the Mupid-2 Mini-Gel System is described. We get sharp and clear bands in Southern and Northern blotting after only 30 min short gel electrophoresis instead of the several hours large gel electrophoresis of conventional methods. The high electrical voltage with a pulse-like current of the Mupid-2 Mini-Gel System also allows reduction of the amount of formaldehyde, a harmful reagent, from the gel running buffer in RNA blotting. This minor modification of DNA and RNA blotting technique enables us to perform the complete experimental procedure more quickly economically in less space, than conventional Southern and Northern blotting, as well as using an extremely small amount of formaldehyde in RNA blotting.     

Abnormal protein patterns in blood serum and cerebrospinal fluid detected by capillary electrophoresis

Capillary zone electrophoresis and high-resolution agarose gel electrophoresis were compared to detect protein components in serum and cerebrospinal fluid of patients. Both electrophoretic methods proved to be useful for detection of protein abnormalities (e.g., mono- and oligoclonal bands) in biological fluids, but capillary electrophoresis offered several important advantages, such as sample application without preliminary concentration, lack of staining procedures, and on-line evaluation of patterns. Furthermore, capillary electrophoresis exhibits shorter analysis time and high resolution with low baseline noise. The results were proven to be powerful in diagnosis and monitoring of dyscrasias in routine laboratory practice.