Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single‐strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA‐based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor‐intensive, time‐consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)‐SSCP/HA. We demonstrate the ability of ME‐SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5–9 in a blinded study in an analysis time of <10 min.     

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.     

Single-strand conformation polymorphism analysis to detect the p53 mutation in colon tumor samples by capillary electrophoresis

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique is developed for the detection of point mutations in DNA samples, and is very useful in the research of tumors. The traditional SSCP was carried out with slab gel electrophoresis (SGE), but this is time-consuming and labor-intensive, particularly for clinical diagnoses. We have developed a capillary electrophoresis (CE) method for SSCP detection with a linear polyacrylamide gel solution as the sieving matrix. Twenty colon tumor samples were detected with SSCP-CE and the point mutation in exon 7 of the p53 gene was found in six of the samples. Based on the sequencing results, the accuracy of SSCP-CE was better than that of SSCP-SGE. We hope this rapid and convenient method could be applied in the clinical diagnosis of tumors soon.     

Applications of homemade kit in mutation detection of genes

With the accomplishment of Human Genome Project (HGP), single nucleotide polymorphism (SNP) and mutation detection in human genome are becom-ing a new researching focus. These researches can help us to understand the phenotype diversity of indi-vidual, disease susceptibility and drug resistance of different colonies. Traditional method used for muta-tion detection is slab gel electrophoresis, which re-mains labor-intensive and time-consuming because of the requirement of radioactivity or te…     

A rapid fluorescence based multiplex polymerase chain reaction--single-strand conformation polymorphism method for p53 mutation detection

We have developed a fast single-strand conformation polymorphism (SSCP) technique to screen for mutations and polymorphisms in exons 5-8 of the human tumor suppressor gene p53. We use multiplex polymerase chain reaction (PCR) to amplify the four exons in one single PCR reaction and then fluorescent SSCP for screening. p53 fragments are labeled with three different colors and a fourth color is used for an internal size marker calibrating the gel. The method was evaluated in two ways: (i) 16 different cell lines with known mutations were tested blindly for band-shifts with SSCP, and (ii) 32 human urinary bladder cancer samples were screened for mutations using the present technique. After screening for mutations all exons from all samples were sequenced, both sense as well as antisense strands. Evaluating the method with four different gels shows that 21/23 mutations and polymorphism were detected in the cell lines and that 10/10 mutations and polymorphisms were detected in the patient samples. Sensitivity, specificity, positive and negative predictive values were 91/100%, 88/ 97%. 78/77% and 96/100% for cell lines / patient samples, respectively. Sensitivity, using one SSCP gel only, was 87% (20/23) for cell lines and 90% (9/10) for patient samples. We conclude that our modified SSCP technique is efficient and has a sensitivity close to 100% in detecting mutations.     

Multitemperature single-strand conformation polymorphism.

Changes of gel temperature during single-strand conformation polymorphism (SSCP) electrophoresis increase the sensitivity of mutation detection in polymerase chain reaction (PCR) products and significantly reduce the overall time and costs of analysis. Based on these findings, a new method for single nucleotide polymorphism (SNP) and point mutation detection--multitemperature single-strand conformation polymorphism (MSSCP) was devised. In order to control the gel temperature with 0.1 degrees C accuracy during electrophoresis, new equipment was developed. We demonstrated that increasing the gel temperature by 8 degrees C or decreasing it by 10 degrees C from 23 degrees C led to the disappearance of all electrophoretic differences between five alleles of exon 8 of the human p53 gene during the SSCP analysis. The interesting result was the detection of two additional SNPs (out of seven analyzed) in exon 7 of the human PAH gene during a one hour MSSCP electrophoresis. This result is better than that obtained by three classical SSCP analyses of the same samples at different but constant gel temperatures. We advocate the MSSCP technology as a fast, reliable, and cost-effective tool for the screening and preselection stage of genomics surveys, especially when a high variability of the analyzed DNA fragment is expected.     

Fluorescent-based single-strand conformation polymorphism/heteroduplex capillary electrophoretic mutation analysis of the P53 gene.

Fluorescent-based single-strand conformation polymorphism (F-SSCP) analysis with capillary electrophoresis (CE) is the most common method for the detection of mutation because of its high sensitivity and resolution. In this study, we prepared an inexpensive linear polyacrylamide (LPA), and successfully applied it to CE-SSCP analysis and tandem CE-SSCP/heteroduplex analysis (HA) of the P53 gene on an ABI capillary genetic analyzer. A comparison of the sieving capabilities of a homemade LPA and commercial polydimethylacrylamide (PDMA) demonstrates that the homemade LPA has a higher resolution, a shorter analysis time, and is more suitable for tandem SSCP/HA than commercial PDMA. To show the usefulness, mutations of P53 gene exon 7 - 8 in 37 tumor samples were investigated by using homemade LPA. The results indicate that 10 mutations were found in 9 of 37 cases; the majority of P53 mutations were missense mutations, and 70% were located in exon 7, which plays an important role in neoplastic progression in human tumorigenesis.     

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.     

Microchip electrophoretic separation for the fast diagnosis of Anaplasma phagocytophilum infection in cattle

We report a diagnostic method for Anaplasma phagocytophilum (A. phagocytophilum) infection in cattle using a nested PCR and microchip electrophoresis (ME). A. phagocytophilum causes human granulocytic anaplasmosis and granulocytic ehrlichiosis, which are emerging tick‐borne zoonotic diseases. Nested PCR was used to amplify genomic DNA samples extracted from cattle blood. The amplified PCR products were analyzed under a sieving gel matrix of 0.7% poly(ethyleneoxide) (M_r=8 000 000) in a conventional glass microchip. In the ME assay, A. phagocytophilum was analyzed within 35 s with a relative standard deviation of 1.30% (n=5) using a programmed field strength gradient (PFSG) as follows: 615.3 V/cm for 0–24 s, 66.7 V/cm for 24–34 s, 615.3 V/cm for 34–100 s. The ME‐PFSG assay was clinically validated by comparing the 16S rRNA gene levels obtained by this method with those measured using conventional slab gel electrophoresis performed with ten cattle blood samples suspected of A. phagocytophilum infection. In contrast to slab gel electrophoresis, the proposed ME‐PFSG methodology had increased sensitivity (200–450 pg/μL), a faster analysis time (<35 s), and required a smaller sample volume (～162 fL).     

Cover Picture: Electrophoresis 21'2011

Issue no. 21 is a regular issue consisting of one FAST TRACK article on “Blinded study determination of high sensitivity and specificity microchip electrophoresis‐SSCP/HA to detect mutations in the p53 gene” and 19 other contributions distributed over 5 distinct parts. Part I is on proteins and proteomics containing 5 contributions describing on‐line combination of multiple chromatographic modes for multicomponent separation of proteomics and glycoproteomics, the heterogeneity of plasma‐derived clotting factor VIII, rapid species evaluation of seafood spoilage and pathogenic Gram‐positive bacteria by MALDI‐TOF mass fingerprinting, comparison of depletion and equalization strategies in proteomics, and a competent extraction method for 2‐D gel electrophoresis. Part II is on affinity capillary electrophoresis and has 2 contributions on the application of partial‐filling using lectins and glycosidases for the characterization of oligosaccharides in therapeutic antibodies, and on protonated diamines as anion‐binding agents and their utility in CE separations. 5 contributions on detection and sensitivity enhancement make up the body of Part III. DNA translocation and depurination constitute the content of the two contributions of Part IV. Finally, Part V assembles 5 papers on various fundamentals and methodologies including the influence of membrane layer properties on the electrophoretic behavior of a soft particle, evaluation of the separation mechanism of EKC with microemulsion and CDs using NMR and molecular modeling, linear polyalkylamines as fingerprinting agents in CE of low‐molecular weight heparins and glycosaminoglycans, use of CE‐SDS gel for the characterization of monoclonal antibody hinge region clipping due to copper and high pH stress, and electrophoretic isolation of saponin fractions from Saponinum album and their evaluation in synergistically enhancing the receptor‐specific cytotoxicity of targeted toxins. In short, this regular issue has a myriad of topics, which make the issue a must to read. Featured articles include: FAST TRACK: Blinded study determination of high sensitivity and specificity microchip electrophoresis‐SSCP/HA to detect mutations in the p53 gene (( 10.1002/elps.201100396 )) Online combination of reversed‐phase/reversed‐phase and porous graphitic carbon liquid chromatography for multicomponent separation of proteomics and glycoproteomics samples (( 10.1002/elps.201100092 )) Trace determination of perchlorate using electromembrane extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection (( 10.1002/elps.201100279 )) Sensitivity enhancement in capillary electrophoresis‐mass spectrometry of anionic metabolites using a triethylamine‐containing background electrolyte and sheath liquid (( 10.1002/elps.201100271 )) Evaluation of the separation mechanism of electrokinetic chromatography with a microemulsion and cyclodextrins using NMR and molecular modeling (( 10.1002/elps.201100263 ))     

微流控芯片电泳激光诱导荧光检测p53外显子上的突变点

利用单链构象多态性(SSCP),建立微流控芯片电泳(ME)联合激光诱导荧光检测(LIF)技术,检测人类p53基因点突变的方法。设计不同碱基长度的p53单链序列,针对易突变的外显子7,8,9进行SSCP分析,分离野生与突变的单链DNA序列;研究了筛分介质聚乙烯基氧化物(PEO)的浓度,场强对芯片电泳行为的影响。在PEO质量分数为0.5%,分离场强为260V/cm时,100 s之内就可以实现样品p53外显子7,8,9的野生型与突变型碱基的分离检测。     

Simultaneous determination of methylephedrine and pseudoephedrine in human urine by CE with electrochemiluminescence detection and its application to pharmacokeinetics

A novel method for the determination of ephedra alkaloids (methylephedrine and pseudoephedrine) was developed by electrophoresis capillary (CE) separation and electrochemiluminesence detection (ECL). The use of ionic liquid (1‐butyl‐3‐methylimidazolium tetrafluoroborate, BMIMBF₄) improved the detection sensitivity markedly. The conditions for CE separation, ECL detection and effect of ionic liquid were investigated in detail. The two ephedra alkaloids with very similar structures were well separated and detected under the optimum conditions. The limits of detection (signal‐to‐noise ratio = 3) in standard solution were 1.8 × 10^–8 mol/L for methylephedrine (ME) and 9.2 × 10^–9 mol/L for pseudoephedrine (PSE). The limits of quantitation (signal‐to‐noise ratio = 10) in human urine samples were 2.6 × 10^?7 mol/L for ME and 3.6 × 10^–7 mol/L for PSE. The recoveries of two alkaloids at three different concentration levels in human urine samples were between 81.7 and 105.0%. The proposed method was successfully applied to the determination of ME and PSE in human urine and the monitoring of pharmacokinetics for PSE. The proposed method has potential in therapeutic drug monitoring and clinical analysis. Copyright © 2009 John Wiley & Sons, Ltd.     

Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis

A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino)ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25 degrees C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2 degrees C/min and were induced externally. A total of 24 samples from affected patients, both in the homo- and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples.     

Voltage‐programming‐based capillary gel electrophoresis for the fast detection of angiotensin‐converting enzyme insertion/deletion polymorphism with high sensitivity

A voltage‐programming‐based capillary gel electrophoresis method with a laser‐induced fluorescence detector was developed for the fast and highly sensitive detection of DNA molecules related to angiotensin‐converting enzyme insertion/deletion polymorphism, which has been reported to influence predisposition to various diseases such as cardiovascular disease, high blood pressure, myocardial infarction, and Alzheimer's disease. Various voltage programs were investigated for fast detection of specific DNA molecules of angiotensin‐converting enzyme insertion/deletion polymorphism as a function of migration time and separation efficiency to establish the effect of voltage strength to resolution. Finally, the amplified products of the angiotensin‐converting enzyme insertion/deletion polymorphism (190 and 490 bp DNA) were analyzed in 3.2 min without losing resolution under optimum voltage programming conditions, which were at least 75 times faster than conventional slab gel electrophoresis. In addition, the capillary gel electrophoresis method also successfully applied to the analysis of real human blood samples, although no polymorphism genes were detected by slab gel electrophoresis. Consequently, the developed voltage‐programming capillary gel electrophoresis method with laser‐induced fluorescence detection is an effective, rapid analysis technique for highly sensitive detection of disease‐related specific DNA molecules.     

Sensitive Determination of 2‐Methoxyestradiol in Pharmaceutical Preparations and Biological Fluids by KMnO4‐Na2SO3 Chemiluminescence System

A simple and novel flow‐injection chemiluminescence (FI‐CL) method was established for the determination of 2‐Methoxyestradiol (2‐ME) in pharmaceutical preparations and biological fluids. The method was based on the significant enhancement of the CL from the KMnO₄‐Na₂SO₃ reaction by 2‐ME in acidic medium. Under optimized conditions, the CL intensity was correlated linearly with concentration of 2‐ME in the range of 5.0 × 10^?8‐5.0 × 10^?6 M (r = 0.9995). The detection limit (3σ) of 2‐ME was 7.5 × 10^?9 M and the relative standard deviation was 0.8% at 5.0 × 10^?7 M 2‐ME (n = 8). The proposed method was successfully applied for the flow‐injection CL determination of 2‐ME in pharmaceutical preparations and biological fluids with the recoveries from 92.4 to 106.8%. The possible CL reaction mechanism was also discussed briefly.     

An Unexpected Spectrum of p53 Mutations from Squamous Cell Carcinomas in Psoriasis Patients Treated with PUVA

Photochemotherapy employing 8-methoxypsoralen and long-wavelength ultraviolet radiation (UVA, 320-400 nm) is widely used in the treatment of psoriasis. The pho-toactivation of psoralens in skin cells leads to formation of DNA photoadducts which may be responsible, at least in part, for the efficacy of these photochemotherapies. However, mutations arising from these adducts may also lead to the well-characterized increased incidence of squamous cell carcinoma. Mutations in the p53 tumor suppressor gene have been detected in many human cancers. To determine whether p53 mutations occur in squamous cell carcinomas in PUVA patients, PCR was used to amplify the exons (5-9) in which other studies have found a high frequency of point mutations. Gel electrophoresis was used to detect single-strand conformational polymorphisms. Aberrantly migrating bands were excised, reamplined and sequenced. Thirty-four specimens from 10 patients were examined. Specimens from one patient who had received no phototherapy as well as from normal controls were also analyzed. Five of the 10 patients showed at least one p53 mutation. In contrast to previously reported psoralen-induced p53 mutations in mice, the expected psoralen type mutations at alternating AT sites were not detected. All but two of the altered sequences occurred at dipyrimidine sites which is typical of solar type mutations. Two C→T mutations and two dipyrimidine mutations (CC→TT) were found. Other mutations included: C→G, G→T, C→A and an 18 bp deletion. A review of therapeutic history of these patients showed that some had also received UVB phototherapy. Furthermore, because sunlight is thought to be beneficial for psoriasis, nontherapeutic, casual UVB exposure cannot be excluded. Our observations suggest that the SCC may have arisen from the solar mutations and that PUVA may enhance tumor progression or immune suppression     

Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.     

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).     

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.     

Use of capillary electrophoresis for high throughput screening in biomedical applications. A minireview

Diagnosis of inherited diseases or cancer predispositions frequently involves determination of specific mutations or polymorphisms. The number of characterized monogenetic and polygenetic diseases is significantly rising every year. As a result, an increasing number of patient samples with a rising complexity of genetic diseases require molecular diagnostics. In order to apply genetic analyses to large groups of patients or population screening, automation of a sensitive and precise method is highly desirable. Capillary electrophoresis (CE) facilitates the development of methods which can rapidly process large number of patient samples in an automated fashion. In contrast, conventional techniques including Southern blotting, sequencing or standard gel electrophoresis are time consuming, cost ineffective and require substantial amounts of each specimen. Robustness, ease of operation, good reproducibility and low cost are the main advantages of CE. Currently, most protocols adapted to automated CE represent (i) analyses of DNA fragment length or DNA restriction patterns (RFLP), (ii) analyses of single-strand conformation polymorphism (SSCP) and (iii) microsatellite analyses. Recently, automated detection of variations in the FRAXA (CGG)n region (fragile X syndrome), LDL receptor gene, p53 gene, MTHFR (methylenetetrahydrofolate reductase) gene, HFE gene and others has been established on CE systems. These applications clearly demonstrate the suitability of CE for high throughput screening in medical applications.