Denaturing gradient-based two-dimensional gene mutation scanning in a polymer microfluidic network

An integrated two-dimensional (2-D) DNA separation platform, combining standard gel electrophoresis with temperature gradient gel electrophoresis (TGGE) on a polymer microfluidic chip, is reported. Rather than sequentially sampling DNA fragments eluted from standard gel electrophoresis, size-resolved fragments are simultaneously electrokinetically transferred into an array of orthogonal microchannels and screened for the presence of sequence heterogeneity by TGGE in a parallel and high throughput format. A bulk heater assembly is designed and employed to externally generate a temporal temperature gradient along an array of TGGE channels. Extensive finite element modeling is performed to determine the optimal geometries of the microfluidic network for minimizing analyte band dispersion caused by interconnected channels in the network. A pH-mediated on-chip analyte stacking strategy is employed prior to the parallel TGGE separations to further reduce additional band broadening acquired during the electrokinetic transfer of DNA fragments between the first and second separation dimensions. A comprehensive 2-D DNA separation is completed in less than 5 min for positive detection of single-nucleotide polymorphisms in multiplex PCR products that vary in size and sequence.     

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).     

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.     

Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis.

Temperature-gradient gel electrophoresis (TGGE) is a technique for studying the structural transitions of nucleic acids and proteins. A temperature gradient is formed in a horizontal slab gel perpendicular to the direction of the electric field. Whereas the principle of the TGGE method has previously been applied to proteins, we describe in this report the systematic optimization of TGGE as a routine technique for the quantitative analysis of conformational transitions in proteins. Using alpha-amylase as an example we show the kinds of results which may be obtained from such measurements. Buffers suitable for use in gel electrophoresis were analyzed with respect to the dependence of their pH value upon temperature. The correct pH range for TGGE of a given protein is determined by electrophoretic titration curves. The protein bands are detected by silver and/or activity staining. The thermal denaturation of alpha-amylase from Aspergillus oryzae showed a discontinuous transition into the denatured conformation, which exhibited much slower electrophoretic mobility. The discontinuity is due to an irreversible denaturation process under the gel conditions. The transition temperature was measured as a function of several parameters, e.g., the concentration of Ca(+)+, dithiotreithol, urea and the pH value. The structural transition of alpha-amylase is accompanied by a loss of enzymatic activity as determined by activity staining or by an activity assay carried out in solution. The structural transitions of two other alpha-amylases from Bacillus subtilis and Bacillus licheniformis were also studied. The results show that the TGGE method is simple to perform and allows the analysis of conformational transitions of proteins in a wide variety of conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Circumventing air bubbles in microfluidic systems and quantitative continuous-flow PCR applications

Polymerase chain reaction (PCR) is an essential part of research based on genomics or cell analysis. The development of a microfluidic device that would be suitable for high-temperature-based reactions therefore becomes an important contribution towards the integration of micro-total analysis systems (μTAS). However, problems associated with the generation of air bubbles in the microchannels before the introduction of the assay liquid, which we call the “initial start-up” in this study, made the flow irregular and unstable. In this report, we have tried to address these problems by adapting a novel liquid-flow method for high-temperature-based reactions. A PDMS-based microfluidic device was fabricated by soft-lithography techniques and placed on a cartridge heater. The generation of the air bubbles was prevented by introducing the fluorinated oil, an inert and highly viscous liquid, as the cap just before the introduction of the sample solutions into the microchannels. The technique was applied for continuous-flow PCR, which could perform PCR on-chip in a microfluidic system. For the evaluation of practical accuracy, plasmid DNA that serves as a reference molecule for the quantification of genetically modified (GM) maize was used as the template DNA for continuous-flow PCR. After PCR, the products were collected in a vial and analyzed by gel electrophoresis to confirm the accuracy of the results. Additionally, quantitative continuous-flow PCR was performed using TaqMan technology on our PCR device. A laser detection system was also used for the quantitative PCR method. We observed a linear relationship between the threshold cycle (Ct) and the initial DNA concentration. These results showed that it would be possible to quantify the initial copies of the template DNA on our microfluidic device. Accurate quantitative DNA analysis in microfluidic systems is required for the integration of PCR with μTAS, thus we anticipate that our device would have promising potential for applications in a wide range of research.     

Quantitative polymerase chain reaction and microchip electrophoresis to detect major rearrangements of the low-density lipoprotein receptor gene causing familial hypercholesterolemia

A variety of rearrangements in the low-density lipoprotein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.     

Electrophoretic mobility of partially denatured DNA in a gel: qualitative and semiquantitative differences between bubbles and split ends

Partially melted DNA is known to exhibit an abrupt decrease of electrophoretic mobility in a gel. Although this is the main phenomenon exploited in TGGE/DGGE (temperature gradient gel electrophoresis/denaturing gradient gel electrophoresis), not much is known about the physical processes responsible for the blocking. While there is a commonly used formula for the reduced mobility based on the theory of branched polymers, it does not discriminate between denatured domains bounded on one (split end) or two sides (bubble). To better understand how the blocking occurs in both of these cases, a coarse-grained model of DNA gel electrophoresis is simulated using Langevin Dynamics. The simulations reveal that the low-field mobility is much more sensitive to denatured domains located at the ends of a DNA fragment. A denatured domain occurring at the center of a fragment indeed reduces the mobility, but at a much lower rate.     

The potential of electrophoretic mobility shift assays for clinical mutation detection

As the understanding of the links between genetic mutations and diseases continues to grow, there is an increasing need for techniques that can rapidly, inexpensively, and sensitively detect DNA sequence alterations. Typically, such analyses are performed on PCR-amplified gene regions. Automated DNA sequencing by capillary array electrophoresis can be used, but is expensive to apply to large numbers of patient samples and/or large genes, and may not always reveal low-abundance mutations in heterozygous samples. Many different types of genetic differences need to be detected, including single-base substitutions and larger sequence alterations such as insertions, deletions, and inversions. Electrophoretic mobility shift assays seem well suited to this purpose and could be used for the efficient screening of patient samples for sequence alterations, effectively reducing the number of samples that must be subjected to full and careful sequencing. While there is much promise, many of the mobility shift assays presently under development have yet to be demonstrated to have the high sensitivity and specificity of mutation detection required for routine clinical application. Hence, further studies and optimization are required, in particular the application of these methods not only to particular genes but also to large numbers of patient samples in blinded studies aimed at the rigorous determination of sensitivity and specificity. This review examines the state-of-the-art of the most commonly used mobility shift assays for mutation detection, including denaturing gradient gel electrophoresis, TGGE, SSCP, heteroduplex analysis, and denaturing HPLC.     

Temperature gradient gel electrophoresis: rapid detection of alpha-1-antitrypsin deficiency carriers.

The homozygous state of the alpha-1-antitrypsin (alpha 1AT) deficiency variant Z is associated with severe liver damage in early childhood and progressive lung emphysema in adulthood. A single base transition (G to A in codon 342) in exon V is causing the severe disease. The Glu342 to Lys342 mutation can be detected conventionally by isoelectric focusing (IEF) or on the DNA level by the newly developed method of temperature gradient gel electrophoresis (TGGE). It is the aim of this study to describe the TGGE technique, to compare the results with conventional IEF, and to discuss its efficiency for different diagnostic applications.     

Towards a portable microchip system with integrated thermal control and polymer waveguides for real-time PCR

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. The integrated polymer optical system for real-time monitoring of PCR was fabricated in the same SU-8 layer as the PCR chamber, without additional masking steps. Two suitable DNA binding dyes, SYTOX Orange and TO-PRO-3, were selected and tested for the real-time PCR processes. As a model, cadF gene of Campylobacter jejuni has been amplified on the microchip. Using the integrated optical system of the real-time PCR microchip, the measured cycle threshold values of the real-time PCR performed with a dilution series of C. jejuni DNA template (2 to 200 pg/microL) could be quantitatively detected and compared with a conventional post-PCR analysis (DNA gel electrophoresis). The presented approach provided reliable real-time quantitative information of the PCR amplification of the targeted gene. With the integrated optical system, the reaction dynamics at any location inside the micro reaction chamber can easily be monitored.     

Advances in molecular techniques for the detection and quantification of genetically modified organisms

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.     

An approach to the sensitivity of temperature-gradient gel electrophoresis in the detection of clonally expanded T-cells in cutaneous T-cell lymphoma

Detection of T-cell clonality by polymerase chain reaction (PCR) and high-resolution electrophoresis facilitates differentiation of early stages of cutaneous T-cell lymphoma (CTCL) from benign T-cell-rich dermatoses. However, data regarding the sensitivity of the various electrophoresis techniques differ remarkably. In the present study, the capacity of heteroduplex (HD)-loaded temperature-gradient gel electrophoresis (TGGE) to detect clonally expanded T-cells was assessed systematically and modifications to the procedure were defined. Using our standard protocol, HD-TGGE detected clonal T-cell receptor (TCR)-gamma PCR products, generated from the Jurkat cell line, down to a total of 2 ng/microL (14 ng) DNA. However, slowly migrating single strands of the clonal PCR product reduced the amount of the clonality indicating homoduplices. To overcome this single-strand formation, thus decreasing the detection limit, the urea concentration in the gel and the temperature ramp for the HD-formation were altered, as well as the temperature gradient in the gel. Application of the modified protocol resulted in a tenfold lower detection limit of 0.15 ng/microL (1.05 ng) DNA in the clonal band. The sensitivity of the adapted HD-TGGE was investigated by dilution experiments using the well established T-cell lines Jurkat, Molt-4, MyLa and SeAx. By these approaches clonal PCR products diluted in nonclonal PCR products were detectable down to concentrations of 5-10%. Comparably, in the case of mixtures of clonal in nonclonal DNA the detection limit reached 5-10% clonal DNA. However, by dilution of clonal cells in nonclonal peripheral blood mononuclear cells, which corresponds to in vivo conditions, a lower detection limit of approximately 1-5% was observed.     

Hundredfold productivity of genome analysis by introduction of microtemperature-gradient gel electrophoresis

Genome profiling, which employs temperature-gradient gel electrophoresis (TGGE) for DNA analysis, has recently been developed in identifying species by genotype. However, the performance of this technology like the general applications of TGGE was, though highly informative, limited in its ability due to methodological reasons. This study demonstrates that minimization of the gel for TGGE, to around one-tenth of its conventional size (approximately 2 cm), can be successfully introduced, resulting in a hundredfold higher performance (total evaluation of time, cost, and degree of parallel operations) than that of the conventional. Reproducibility was evaluated from the measures of the pattern similarity scores (PaSS) between band patterns (genome profiles) obtained with the conventional TGGE, and that with micro-TGGE (microTGGE) developed here, after extracting a set of featuring points from genome profiles. Size minimization, which leads to the reduction of the amount of samples required (cost-saving), is another great advantage, enhancing the employment of multicolor fluorescence technology. Since the further development of microbe-related fields such as epidemiology and microbial ecology inevitably require knowledge based on the identification of a great number of species and strains, microbe-related fields will receive the most optimal benefits from the technological improvements attained here.     

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.     

Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency

Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) has been developed to detect polymerase chain reaction (PCR) amplified samples. LIF detection was performed using Thiazole Orange as the fluorescent intercalating dye. This method was ca. 100X as sensitive as that with UV detection. The highly sensitive CGE-LIF was applied to the detection of the most prevalent mutation (lysine³²⁹- to-glutamic acid substitution) in medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The disorder, which shows an autosomal recessive inheritance, is known to be highly prevalent among Caucasian population and often mimics as Reye-like syndrome or sudden infant death.

A DNA fragment containing the mutation site was PCR-amplified with two sets of allele specific oligonucleotide primers, followed by CGE-LIF. The mutant allele produced a 175-base pairs DNA fragment, which the normal allele generated a 202-base pairs DNA fragment. CGE-LIF clearly distinguished these PCR products, facilitating rapid diagnosis of MCAD deficiency.     

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.     

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.     

Integration of combined heteroduplex/restriction fragment length polymorphism analysis on an electrophoresis microchip for the detection of hereditary haemochromatosis

This work describes an integrated method of enzymatic digestion, heteroduplex analysis (HA) and electrophoretic sizing on a microfluidic chip. HA techniques based on microchip electrophoresis are capable of the high sensitivity detection of subtle mutations such as single nucleotide polymorphisms (SNPs) but are not readily able to detect homozygous mutant genotypes. Such homozygous conditions are commonly encountered with the gene implicated in hereditary haemochromatosis, HFE. We employed the restriction fragment length polymorphism (RFLP) method of mutation detection to complement the HA method in a rapid novel on-chip procedure that separated digested PCR fragments to reliably determine the presence or absence of the most important mutations associated with haemochromatosis. This method was able to distinguish the homozygous mutant, heterozygous and homozygous wildtype genotypes. The mutations investigated here (C282Y, H63D and S65C) are often the mutation targets used in the genetic testing for haemochromatosis. This method provides the extremely specific digestion methods needed for the analysis of the known and relatively common mutations that have a significant probability of occurring in a homozygous form. However, the high sensitivity of the HA method is useful in detecting other mutations of lesser likelihood which, by virtue of their rarity, are likely to be present only in a heterozygous form. Although the conventional methods of analysing these mutations require as much as a day to perform, this microchip method, even without robotics or multiplexed operation, can be performed in about 10 min per sample.     

Gel immobilization of acrylamide-modified single-stranded DNA template for pyrosequencing

A novel two-step process was developed to prepare ssDNA templates for pyrosequencing. First, PCR-amplified DNA templates modified with an acrylamide group and acrylamide monomers were copolymerized in 0.1 M NaOH solution to form polyacrylamide gel spots. Second, ssDNA templates for pyrosequencing were prepared by removing electrophoretically unbound complementary strands, unmodified PCR primers, inorganic pyrophosphate (PPi), and excess deoxyribonucleotides under alkali conditions. The results show that the 3-D polyacrylamide gel network has a high immobilization capacity and the modified PCR fragments are efficiently captured. After electrophoresis, gel spots copolymerized from 10 microL of the crude PCR products and the acrylamide monomers contain template molecules on the order of pmol, which generate enough light to be detected by a regular photomultiplier tube. The porous structure of gel spots facilitated the fast transportation of the enzyme, dNTPs and other reagents, and the solution-mimicking microenvironment guaranteed polymerase efficiency for pyrosequencing. Successful genotyping from the crude PCR products was demonstrated. This method can be applied in any laboratory; it is cheap, fast, simple, and has the potential to be incorporated into a DNA-chip format for high-throughput pyrosequencing analysis.