High-throughput genotyping of factor V Leiden mutation by ultrathin-layer agarose gel electrophoresis.

Ultrathin-layer agarose gel electrophoresis is a novel combination of the established methodologies of slab gel electrophoresis and capillary gel electrophoresis. This new format provides a multilane separation platform with rapid analysis time and excellent sensitivity by using laser-induced fluorescence scanning detection system. Sample injection onto the ultrathin-layer separation platform is easily accomplished by membrane mediated loading technology. In this paper, we demonstrate the sensitivity and high-throughput fashion of this novel separation and detection system for rapid genotyping of the coagulation factor V Leiden mutation by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis. The PCR amplified fragment from exon 10 of the factor V gene was digested by the Mnl I restriction enzyme, followed by automated ultrathin-layer agarose gel electrophoresis analysis with "in migratio" fluorescent labeling during the separation process. Due to its speed and automation, this method should be considered for large scale screening of factor V Leiden mutation.     

Efficient mutation detection in MEN1 gene using a combination of single-strand conformation polymorphism (MDGA) and heteroduplex analysis

For facilitated genotypic analysis of multiple endocrine neoplasia type 1 (MEN1), a familial syndrome associated with tumors of the parathyroid and neuroendocrine tissues, we developed two screening methods, heteroduplex mutation assay (HMA) and mutation detection gel analysis (MDGA), both based on electrophoretic discrimination of polymerase chain reaction (PCR) products, to detect the mutations. Forty-three genomic DNA samples were used for the evaluation of these techniques. The whole coding region of MEN1 was PCR-amplified with fluorescent primers and then denatured/renatured before electrophoresis on an automated sequencer. 100% of the mutations were detected, subsequently confirmed and identified by sequencing. "Negative" samples were used to evaluate the specificity and reproducibility of the two techniques. The combination of the two methods allows high throughput cost-effective mutation screening which is less laborious than systematic sequencing of the whole coding region of MEN1. Together, these methods provide an efficient screen for MEN1 mutations.     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele-specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium-bromide-labeled fragments.     

Detection of CFTR mutations using temporal temperature gradient gel electrophoresis

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is one of the most common autosomal recessive diseases with variable incidences and mutation spectra among different ethnic groups. Current commercially available mutation panels designed for the analysis of known recurrent mutations have a detection rate between 38 to 95%, depending upon the ethnic background of the patient. We describe the application of a novel mutation detection method, temporal temperature gradient gel electrophoresis (TTGE), to the study of the molecular genetics of Hispanic CF patients. TTGE effectively identified numerous rare and novel mutations and polymorphisms. One interesting observation is that the majority of the novel mutations are splice site, frame shift, or nonsense mutations that cause severe clinical phenotypes. Our data demonstrate that screening of the 27 exons and intron/exon junctions of the CFTR gene by TTGE greatly improves the molecular diagnosis of Hispanic CF patients.     

BRAF mutation detection and identification by cycling temperature capillary electrophoresis

BRAF mutations are found in many human tumors, namely melanomas ( approximately 70%) and colon carcinomas ( approximately 15%). This paper presents a method for identification of exon 15 BRAF mutations by denaturant capillary electrophoresis (CE), an analysis method that is sensitive, cost-effective (involving only polymerase chain reaction (PCR) and electrophoresis) and capable of high-throughput screening. In total, we found 21 (70%) out of 30 melanoma cell lines with BRAF mutations in exon 15: two of which were the p.Val600Asp (c.1799-800TG>AT) mutation, one cell line contained the p.Val600Arg (c.1798-99GT>AG) mutation, and 18 cell lines contained the p.Val600Glu (c.1799T>A) mutation. Of the nine cell lines that did not contain a BRAF mutation, five contained an NRAS mutation at exon 2, and no mutations were detected in NRAS exon 1. There was no overlap of NRAS and BRAF mutations in the same cell line. In addition, we looked at 221 colon biopsy samples and identified one further BRAF mutation, the p.Asp594Gly (c.1781A>G) mutation, in seven samples. The p.Val600Glu mutation was identified in 11 of the colon biopsy samples. Using the four mutations of BRAF exon 15, we then constructed a denaturing CE standard capable of distinguishing between each of the mutations; therefore, sequencing does not need to be performed to confirm the mutation. In conclusion, this sensitive, cost-effective mutation assay for BRAF (and RAS) will provide the opportunity to detect and determine mutations without the need to purify samples for sequencing. Future large-scale studies will provide the clinical usefulness of such mutations.     

Glycerol-enhanced mini-polyacrylamide gel electrophoresis for the separation of differentially expressed DNA fragments in cDNA representational difference analysis

Representational difference analysis (RDA) is a widely used technique in molecular biology. However, in practice, its efficiency depends on a rapid and reliable separation of the RDA fragments prior to cloning. To achieve this, we have compared and combined the separation efficiencies of conventional and MetaPhor agarose gel electrophoresis (MAGE) with a glycerol-enhanced mini-polyacrylamide gel electrophoresis (PAGE) system (Gem-PAGE). As anticipated, MetaPhor agarose provided significantly improved resolution over conventional agarose electrophoresis, but the latter remains useful to rapidly confirm the presence of RDA-enriched difference products and direct the concentration of MetaPhor agarose subsequently used for further fragment separation. Additional improvements in resolution were possible by using the Gem-PAGE system. The effect of glycerol on band definition of PAGE was most noticeable as the acrylamide to glycerol ratio (A:G) approached 1:2. Gels in which the A:G ratio was significantly above or below this resulted in both poor morphology and impaired resolution of the bands. By exploiting sequentially agarose-based and Gem-PAGE electrophoresis, the goal in RDA of "one band one product" is now realizable.     

A modified method of genomic DNA preparation in agarose inserts for pulse field gel electrophoresis.

According to standard protocol, DNA in agarose inserts is prepared by first embedding the cell in agarose. This is then incubated in the required enzyme (lysozyme, lysostaphin, or zymolase) depending on the cell type (bacterial or plants), for spheroplast formation. Subsequent treatment of the spheroplast with proteinase K allows the isolation of large genomic DNA in agarose suitable for pulse field gel electrophoresis. An efficient and rapid method of preparation of spheroplast is described. In this method a low concentration of enzyme required for spheroplast formation was added before embedding the cell in agarose, which facilitated the digestion of cell wall by the enzyme and allowed use of a low amount of enzyme. Digestion of DNA in agarose inserts prepared by this method, with rare cutting restriction enzyme and pulse field gel electrophoresis, showed that the quality of DNA was as good as obtained by the standard method.     

Factor B (BF) subtyping by isoelectric focusing: methods, nomenclatures, genetics and forensic application

Usually factor B (BF) typing is performed by means of the traditional agarose gel electrophoresis. Using isoelectric focusing, the system can be extended by two common subtypes of BF F. The existence of BF F subtypes has in the meantime been confirmed by various authors and in different populations. Their inheritance has been proven by family- and mother/child analyses and molecular-genetic studies (correlation with restriction fragment length polymorphism). Different typing methods as well as different nomenclatures seem to indicate that the subtypes FA and FB (according to Geserick et al.) are identical with the Fb and Fa subtypes (according to Teng and Tan). At present, some confusion still exists for the less frequent variants and subtypes which possibly could be identified by direct comparison of the patterns. The BF system is a valuable marker in paternity testing. Its chance for exclusion of paternity in Caucasian populations has been calculated to be about 14% for agarose gel electrophoresis and increases to about 16% for BF F subtyping by isoelectric focusing. Preliminary results indicate that BF may also be used for typing of bloodstains (up to 2 weeks old).     

Use of denaturing gradient gel electrophoresis to detect mutation in VS2 of the 16S-23S rDNA spacer amplified from Staphylococcus aureus isolates

To develop a double gradient denaturing gradient gel electrophoresis (DG-DGGE) based typing method that rapidly and accurately types clinical isolates of Staphylococcus aureus, the VS2 region of the 16S-23S rRNA spacer region (ISR) was chosen because of its potential high variation. The VS2 region was amplified with a 40-mer GC-clamp attached to the 5'-end of the reverse primer. The 145 bp PCR product was then separated by DG-DGGE using denaturant concentrations of 25-40% and polyacrylamide concentrations of 6-12%. Of the five mutations identified in 336 S. aureus isolates, one mutation was found to be highly specific for 161/171 (94%) of methicillin-resistant S. aureus (MRSA) isolates from different geographic locations and isolation times. This same mutation was found in 15/160 (9%) of penicillin- or methicillin-sensitive S. aureus isolates. In some isolates two mutations occured together in the one genome suggesting some S. aureus isolates have two copies of VS2. In these 336 isolates nine genotypes with different combinations of the five mutations were identified. In 18 coagulase-negative staphylococci (CNS), the MRSA-specific mutation was found along with two other mutations in all isolates demonstrating consistent differences in the presence of these mutations between CNS and S. aureus. The marked differences in VS2 sequences found between MRSA, methicillin- or penicillin-sensitive S. aureus (SSA), and CNS by DGGE in the present study may be useful in evolutionary studies and in the development of a specific assay for MRSA from clinical specimens.     

Development of a novel two-dimensional gel electrophoresis protocol with agarose native gel electrophoresis

A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining the recently developed agarose native gel electrophoresis with either vertical sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. Our innovative technique utilizes His/MES buffer (pH 6.1) during the first-dimensional (1D) agarose native gel electrophoresis, which allows for the simultaneous and clear visualization of basic and acidic proteins in their native states or complex structures. Our agarose gel electrophoresis is a true native electrophoresis, unlike blue native–PAGE, which relies on the intrinsic charged states of the proteins and their complexes without the need for dye binding. In the 2D, the gel strip from the 1D agarose gel electrophoresis is soaked in SDS and placed on top of the vertical SDS–PAGE gels or the edge of the flat SDS–MetaPhor high-resolution agarose gels. This allows for customized operation using a single electrophoresis device at a low cost. This technique has been successfully applied to analyze various proteins, including five model proteins (BSA, factor Xa, ovotransferrin, IgG, and lysozyme), monoclonal antibodies with slightly different isoelectric points, polyclonal antibodies, and antigen–antibody complexes, as well as complex proteins such as IgM pentamer and β-galactosidase tetramer. Our protocol can be completed within a day, taking approximately 5–6 h, and can be expanded further into Western blot analysis, mass spectrometry analysis, and other analytical methods.     

High-throughput screening of genetic mutations/polymorphisms by capillary electrophoresis

Genetic mutations/polymorphisms analyses play a great role in genetic and medical research, and clinical diagnosis. Most conventional methods for genetic assay are based on slab gel electrophoresis that is both labor-intensive and time-consuming. Recently, capillary electrophoresis (CE) has been used for genetic analysis instead of conventional slab gel electrophoresis. This technique can be automated and is characterized by short analysis time, small sample and reagents requirements, and high separation efficiency. CE has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes, and has shown a great potential for genetic mutation/polymorphism screening of large numbers of clinical samples. In this article, an overview of the fundamental aspects of mutation/polymorphism assay methods in combination with CE is given and some key applications are summarized.     

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.     

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis without a stacking gel: use for high sample throughput

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis without a stacking gel minimizes lateral spreading of protein when samples are applied in agarose wells and allows high sample throughput (6 samples/cm gel width). The method is simple and convenient to use and gives comparable resolution to the standard method with 4-20% or 6-30% polyacrylamide gradient gels. Best results are obtained when the upper zone of the separating gel is of low polyacrylamide concentration. This indicates a need for the molten agarose to penetrate and anneal with the separating gel.     

Temperature-gradient gel electrophoresis for the detection of polymorphic DNA and for quantitative polymerase chain reaction.

In order to detect mutations in a gene, either known mutations from human diseases or artificial ones in transgenic animals, or to screen for not yet identified mutations in patients, a method is required which guarantees detection of mutations which might occur in every single position of the whole open reading frame (ORF). It will be shown that a combination of polymerase chain reaction (PCR) and temperature gradient gel electrophoresis (TOGE) fulfills these requirements. By thermodynamic calculations the shift in the gel electrophoresis due to a mutation can be calculated in dependence on the position of the mutation. The theoretical results were tested with the mutations known so far. The quantitative determination of the copy number of a specific DNA or RNA sequence in a biological specimen (quantitative PCR) can be performed precisely and easily by combining PCR and TGGE. The system uses a quantification strategy of a new type of internal standardization. TGGE is applied to separate homo- and heteroduplexes which correspond respectively to standard and template sequences. The accuracy of this quantification strategy is very high, with a variability of < 15%. In addition to quantification, PCR/TGGE detects PCR artifacts and template mutants.     

Preparation of plant DNA for separation by pulsed field gel electrophoresis

A method was developed for the preparation of completely intact plant DNA embedded in agarose, and suitable for restriction enzyme digestion. Digestion with restriction enzyme was carried out according to modified protocols of Anand and Kenwrick et al. The new method of DNA isolation allows the separation of high molecular weight plant DNA by pulsed field gel electrophoresis.     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

High throughput mutation screening by automated capillary electrophoresis

Molecular diagnosis of complex inherited disorders, population screening of genetic diseases, studies of the genetic basis of variable drug response (pharmacogenetics) as well as discovery and investigation of new drug targets (pharmacogenomics) involve screening for mutations in multiple DNA samples. Furthermore, the development of a third generation of the human genome map, based on single nucleotide polymorphisms (SNPs), requires screening for allelic variants through all of the three billion basepairs in the human genome. Thus, the need for high throughput mutation screening methods is great and is rapidly increasing. Traditional methods for mutation screening often involve slab-gel electrophoresis analyses which are laborious and difficult to automate. However, recent developments in capillary electrophoresis systems for DNA fragment analysis have made fully automated mutation screening possible and have dramatically increased the possible sample throughput. This review describes the recent advances in capillary electrophoresis of DNA and summarize the various methods for mutation screening based on this technique.