Design and application of 2-D DGGE-based gene mutational scanning tests

Currently, there is a need for practical, accurate and cost-efficient tests to comprehensively scan human genes for disease-related DNA sequence variation. Two-dimensional gene scanning (TDGS) is a parallel mutation detection system, based on a combination of extensive multiplex PCR amplification (‘PCR megaplex’) and two-dimensional (2-D) DNA electrophoresis. The latter comprises a size separation step followed by denaturing gradient gel electrophoresis (DGGE), and allows single base pair changes to be distinguished among multiple DNA fragments in parallel. Here, we describe the rapid design of TDGS tests and its application to mutation identification in several large human cancer genes.     

Mutation screening using fluorescence multiplex denaturing gradient gel electrophoresis (FMD): detecting mutations in the BRCA1 gene

Fluorescent multiplex denaturing gradient gel electrophoresis (FMD) is a mutation screening technique designed to detect unknown as well as previously identified mutations. FMD constitutes a recent modification of the standard denaturing gradient gel electrophoresis (DGGE) technique, which combines multiplex PCR amplification of target DNA using fluorescently labeled primers with DGGE separation of the amplicon mixture, allowing immediate identification of sequence variants by wet gel scanning. FMD permits the simultaneous detection of small insertions, deletions and single nucleotide substitutions among multiple DNA fragments (up to 480 fragments) from 96 samples in parallel for each run. It increases output and reduces cost dramatically compared with classical DGGE, without sacrificing sensitivity and accuracy in detecting mutations. This protocol details an accurate, fast, nonradioactive and cost-effective way to screen the BRCA1 gene for mutations with high sensitivity, providing easily interpreted results. It may also be adapted to screen other target genes and/or used in large-scale epidemiological studies.     

The isolation of CpG islands from human chromosomal regions 11q13 and Xp22 by segregation of partlymelted molecules.

We isolated fragments containing parts of CpG islands from human chromosomal regions chosen for expected differences in gene density by segregation of partly melted molecules. Restriction fragments of P1 bacteriophage clones covering a region of 11q13 and those of cosmid clones derived from Xp22 were recovered from bands in denaturing gradient gels that were retained following prolonged exposure to electric field. Forty-five independent fragments derived from 11q13 and five from Xp22 were isolated. Nucleotide sequence analysis revealed that 11 of the 45 fragments from 11q13 contained CpG islands including four derived from known genes in 11q13. None of the five fragments derived from Xp22 resembled CpG islands. The number of CpG island fragments obtained was consistent with the expectation based on the number of Not I restriction endonuclease sites present at these regions. Adjustment of parameters in our quasi-theoretical approach to the rate of fragment dissociation improves the discrimination between retention and non-retention. The results support probable identification of CpG island fragments by their reduced rate of strand dissociation when retarded in a denaturing gradient gel.     

Denaturing high-performance liquid chromatography is a suitable method for PMM2 mutation screening in carbohydrate-deficient glycoprotein syndrome type IA patients

The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065). The gene spans 8 exons and 741 bp of coding DNA. Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon. The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degrees C with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.     

Comprehensive, rapid and sensitive detection of sequence variants of human mitochondrial tRNA genes.

In the present study, a comprehensive, rapid and sensitive method for screening sequence variation of the human mitochondrial tRNA genes has been developed. For this purpose, the denaturing gradient gel electrophoresis (DGGE) technique has been appropriately modified for simultaneous mutation analysis of a large number of samples and adapted so as to circumvent the problems caused by the anomalous electrophoretic behavior of DNA fragments encoding tRNA genes. Eighteen segments of mitochondrial DNA (mtDNA), each containing a single uniform melting domain, were selected to cover all tRNA-encoding regions using the computer program MELT94. All 18 segments were simultaneously analyzed by electrophoresis through a single broad range denaturing gradient gel under rigorously defined conditions, which prevent band broadening and other migration abnormalities from interfering with detection of sequence variants. All base substitutions tested, which include six natural mutations and 14 artificially introduced ones, have been detected successfully in the present study. Several types of evidence strongly suggest that the anomalous behavior in DGGE of tRNA gene-containing mtDNA fragments reflects their tendency to form temporary or stable alternative secondary structures under semi-denaturing conditions. The high sensitivity of the method, which can detect as low as 10% of mutant mtDNA visually, makes it valuable for the analysis of heteroplasmic mutations.     

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE)

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.     

DDGGE, a simplified denaturing gradient gel electrophoresis method for the detection of sequence variations in PCR fragments

The need for rapid analysis of sequence variations in PCR fragments of the same length is increasing in medical diagnostics and environmental studies. Therefore a modified denaturing gradient gel electrophoresis (DGGE) method was developed in which mixed PCR fragments of 1,500 bp could be analysed on a conventional DNA sequencing gel apparatus. In addition, PCR primers without long GC-clamps could be used to amplify the target genes. © Rapid Science Ltd. 1998     

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated. Electronic Publication     

An efficient screening procedure detecting six novel mutations in the LDL receptor gene in Swedish children with hypercholesterolemia

Familial hypercholesterolemia (FH) is an autosomal semi-dominant disorder caused by defects in the low density lipoprotein receptor (LDLR) gene and is a well-documented risk factor for developing cardiovascular disease. The LDLR genes of five Swedish children with FH were examined in this study. Initial mutation screening was performed by denaturing gradient gel electrophoresis (DGGE) with enzymatically amplified exon-sized fragments, each containing a tailing GC-rich requence. The GC-clamped fragments had been synthesized with a restriction site adjacent to the intron-corresponding sequence to allow detachment of the clamps, thereby rendering the fragments suitable for subsequent analysis by single-strand conformation polymorphism (SSCP) analysis of samples from patients with no DGGE-detectable mutations. In addition, all the LDLR genes of the patients were screened for large alterations by restriction fragment length polymorphism analysis. Following this strategy, seven different, potentially disease-causing mutations were detected in the five children with FH. Six of the alterations, five single-base substitutions and one dinucleotide deletion, have not previously been described. DGGE detected six of the mutations and SSCP the seventh.     

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.     

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here.This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.     

Construction of a chromosome 15-specific linking library and identification of potential gene sequences

We have tested a strategy for the construction of a total restriction fragment map of a human chromosome and the rapid isolation of a great number of genes from a specific chromosome. The strategy is based on the cloning of chromosome-specific CpG-rich DNA sequences which are present at the 5' end of many genes. This approach has two important implications: (i) the clones can be used to probe pulsed-field gradient gel blots and link restriction fragments generated by CpG restriction endonucleases, and (ii) the finding of tight genetic or physical linkage between one of these gene probes and a given hereditary disease would make the marker a good candidate gene for this disease. We have constructed a chromosome 15-specific linking library and identified potential gene sequences.     

Functional adaptation of microbial communities from jet fuel-contaminated soil under bioremediation treatment: simulation of pollutant rebound

To investigate the link between the functionality and the diversity of microbial communities under strong selective pressure from pollutants, two types of mesocosms that simulate natural attenuation and phytoremediation were generated using soil from a site highly contaminated with jet fuel and under air-sparging treatment. An increase in the petroleum hydrocarbon concentration from 4900 to 18,500 mg kg(-1) dw soil simulated a pollutant rebound (postremediation pollutant reversal due to residual contamination). Analysis of soil bacterial communities by denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments showed stronger changes and selection for a phylogenetically diverse microbial population in the mesocosms with pollutant-tolerant willow trees. Enumeration of the main subfamilies of catabolic genes characteristic to the site detected a rapid increase in the degradation potential of both systems. A marked increase in the abundance of genes encoding extradiol dioxygenases with a high affinity towards various catecholic substrates was found in the planted mesocosms. The observed adaptive response to the simulated pollutant rebound, characterized by increased catabolic gene abundance, but with different changes in the microbial structure, can be explained by functional redundancy in biodegrading microbial communities.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Molecular Analysis of Recombination Events in Drosophila

The locations of crossover junctions and gene conversion tracts, isolated in the rosy gene of Drosophila melanogaster, were determined using DNA sequencing and denaturing gradient gel electrophoresis. Frequent DNA sequence polymorphisms between the parental genes served as unselected genetic markers. All conversion tracts were continuous, and half of the reciprocal crossover events had conversion tracts at the crossover junction. These experiments have also identified the sequence polymorphisms responsible for altered gene expression in two naturally occurring rosy variants.     

Mutation analysis of p53 in ovarian tumors by DHPLC

Up to now, ovarian carcinomas represent a major health problem among female cancers because they are the leading cause of death from gynecological malignancy. A high proportion of these tumors selects for mutations in the p53 gene. There is evidence that inactivation of the p53 protein could indicate poor prognosis and chemoresistance of patients. To set up a fast and sensitive test for p53 defects in tumor tissues, we analyzed ovarian cancer cells by denaturing high-performance liquid chromatography (DHPLC). A primer set spanning the whole coding region of p53 with seven fragments was designed and appropriate heteroduplex detection in DHPLC analysis was elaborated. The analysis of 45 ovarian tumor specimens yielded 17 genetic alterations (38%) occurring exclusively in the malignant tissue of the patients. In addition, frequent polymorphisms present in normal compared to tumor tissue could serve as a tool for the rapid identification of loss of heterozygosity (LOH) in the tumor. We observed that LOH in intron 2 or 3 correlated well with a lack of one allele in mutated fragments. In conclusion, DHPLC screening appears to be a sensitive and effective test for genetic alterations in tumors with p53 involvement. Since p53 mutations point to a poor prognosis state in several cancers, a fast screening of tumor material for genetic variations may have implications for further individual treatment of patients.     

Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments.

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.     

Genetic Diversity of cry Gene Sequences of Bacillus thuringiensis Strains Analyzed by Denaturing Gradient Gel Electrophoresis

PCR has been widely used to identify cry-type genes, to determine their distribution, to detect new such genes and to predict insecticidal activities. We describe here a molecular approach to analyze the genetic diversity of B. thuringiensis cry-like genes based on denaturing gradient gel electrophoresis (DGGE). This analysis demonstrated that different B. thuringiensis isolates can be distinguished according to its PCR-DGGE profile of cry-like genes. Identification of the resolvable DNA fragments was easy to accomplish by DNA sequencing, which was confirmed in this work. Importantly, the strategy allowed the identification of unknown B. thuringiensis cry-like sequences present in a single strain that remained cryptic after PCR analysis using degenerate primers. The method developed in this work contributes to the availability of molecular techniques for both B. thuringiensis strains and cry-like genes identification and discovery.     

Yeast species recognition from gene sequence analyses and other molecular methods

This review discusses DNA-based methods used for identification of yeasts. Nuclear DNA reassociation was the first quantitative molecular method employed for recognition of yeast species and has provided a baseline for interpretation of other molecular comparisons. Among these, gene sequencing is the most definitive method, with ribosomal RNA gene sequences providing the preponderance of available data. Multigene analyses that include the sequences of protein encoding genes are being increasingly developed to provide a more definitive resolution of species. A number of rapid identification methods, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and flow cytometry, which are based on species-specific gene sequences, are available for use in diagnostic laboratories.     

Genetic variation in the porcine myogenin gene locus

The myogenin (MYOG) gene fulfills a key function in muscle differentiation by controlling the onset of myoblast fusion and the establishment of myofibers. In meat-producing animals like pigs and cattle, myofiber numbers have been related to growth capacity. We have characterized the porcine MYOG gene to detect genetic variation at this locus and to relate it to growth characteristics. MYOG gene fragments were isolated by PCR on genomic DNA and by screening a genomic library with a mixture of the four human MyoD cDNA fragments. Both the exons and promoter region were very similar to the human and mouse genes. Southern blot analysis of 105 unrelated pigs revealed three polymorphic MspI sites, located in the promoter region, the second intron, and at the 3′ side of the gene. PCR-RFLP tests detecting four MYOG alleles were developed. PCR analysis of a panel of pig–rodent somatic cell hybrids confirmed the genetic localization of MYOG on pig Chromosome (Chr) 9. The PCR-RFLP tests and microsatellite markers on Chr 9 offer the possibility to genotype large numbers of pigs for studies of genetic linkage to meat deposition and growth characteristics. Received 13 October 1996 / Accepted: 16 March 1992