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.     

Angiotensin I-converting enzyme (ACE): estimation of DNA haplotypes in unrelated individuals using denaturing gradient gel blots

The angiotensin I-converting enzyme (ACE) gene (17q23) is a candidate gene for essential hypertension and related diseases, but investigation of its role in human pathology is hampered by a lack of identified polymorphisms. Currently, a 287-bp insertion/deletion (I/D) RFLP in intron 16 represents the only one known. Additional polymorphisms for the ACE gene would make most families informative for linkage studies and would allow haplotypes to be assigned in association studies. To increase the information provided by the ACE gene, we used a sensitive screening technique, denaturing gradient gel electrophoresis (DGGE) blots, to identify polymorphisms and combined this with gene counting to identify haplotypes. Five independent polymorphisms, restriction fragment melting polymorphisms (RFMPs), were identified by four probes (encompassing half of the ACE cDNA) in digests produced by three restriction enzymes (DdeI, RsaI, and AluI). One RFMP has three alleles while the others have two alleles. In a sample of 67 unrelated control subjects, minor allele frequencies ranged from 0.12 to 0.49. A significant level of linkage disequilibrium was found for all pairs of markers. The four most informative RFMPs, taken in combination, define 24 potential haplotypes. Based on gene counting, 11 of the 24 are rare or nonexistent in this population, and the estimated heterozygosity of the remaining 13 haplotypes approaches 80%. Under these conditions for the ACE locus, phase-unknown genotypes could be assigned to haplotype pairs in unrelated subjects with reasonable certainty. Thus, using DGGE blot technique for identifying numerous DNA polymorphisms in a candidate locus, in combination with gene counting, one can often identify DNA haplotypes for both related and unrelated study subjects at a candidate locus. These markers in the ACE gene should be useful for clinical and epidemiologic studies of the role of ACE in human disease.     

Syndromes d’insensibilite aux androgenes: Aspects moleculaires

The molecular analysis of clinical syndromes involving androgen insensitivity has been facilitated by the availability of increasingly powerful molecular biological techniques. Complementary DNA of the androgen receptor gene (wich was recently cloned and sequenced) has been used as a probe to investigate DNA restriction fragment length polymorphisms in patients with partial or complete androgen insensitivity. Such studies have demonstrated that deletions are rare. Using enzymatic amplification and sequencing of exons of the androgen receptor gene, several groups have described point mutations in patients with androgen insensitivity. PCR, accompanied by denaturing gradient gel electrophoresis or single strand conformation polymorphism assays, has revealed further mutations. These powerful tools, together with studies of mRNA from expression of the mutant gene, have also illustrated structure-function associations of the androgen receptor gene in some patients with androgen insensitivity.     

A fluorescent multiplex-DGGE screening test for mutations in the BRCA1 gene

Screening for mutations in the BRCA1 gene is challenging because of the wide spectrum of mutations found in this large gene. As the extensive exon 11 is commonly screened by the protein truncation test (PTT), here a fluorescent multiplex denaturing gradient gel electrophoresis (FMD) mutation screening technique was developed to test the remaining numerous small exons and splice sites of the gene. The method is based upon the use of an efficient multiplex polymerase chain reaction (PCR) amplification of the target regions, followed by denaturing gradient gel electrophoresis (DGGE) separation of the amplicon mixture, and the immediate achievement of results by wet gel scanning. The technique was applied to screen 16 samples with different BRCA1 sequence variants distributed over 12 exons. All variants were detected. In addition, 188 DNA samples from ovarian cancer patients were screened, identifying 22 new sequence variants (11.7% of the samples) and 243 common polymorphisms in the BRCA1 locus. Variants included 16 single nucleotide substitutions, 3 deletions of 2 nucleotides, 1 deletion of 4 nucleotides, and 2 insertions of 1 nucleotide. The FMD test provides an accurate, fast, nonradioactive and cost-efficient way to scan the BRCA1 gene with high sensitivity and an ease of result interpretation. This technique may prove to be a useful research tool for the detection of mutations and polymorphisms in the BRCA1 gene and for large-scale epidemiologic studies.     

Intramolecular DNA melting between stable helical segments: melting theory and metastable states.

Melting of DNA in a segment bounded at both ends by regions of greater stability during electrophoresis in denaturing gradient gels show complex properties, not accommodated with standard melting theory. Compact bands of some DNA molecules become anomalously broadened at the retardation level in a denaturing gradient, or double bands may appear in a uniform denaturant concentration. These properties are associated only with molecules for which the distribution of stability calculated by the Poland-Fixman-Freire algorithms indicates that the region of lowest stability does not extend to an end of the molecule. Retention of helicity at the ends is shown by the difference in the effect of base substitution in the end domains and in the least stable domain. Both the appearance of double bands and band broadening can be explained by invoking a hypothetical metastable intermediate in melting, which is converted into the equilibrium melted form at a relatively slow rate, depending on both denaturant concentration and field strength. A kinetic model permits plausible rate constants to be inferred from the patterns. Despite the increased band width, sequence variants with base changes in the least stable domain result in readily detectable band shifts in the gradient.     

Detection of multiallele polymorphisms within gene sequences by GC-clamped denaturing gradient gel electrophoresis.

The ability to efficiently detect DNA polymorphisms is essential for the completion of a high-resolution polymorphic linkage map of the human genome. Currently the most informative polymorphisms are the multiallelic dinucleotide repeat polymorphisms. However, many gene sequences lack an associated dinucleotide repeat sequence. We used GC-clamped denaturing gradient gel electrophoresis to screen for DNA polymorphisms in the following six gene sequences: MCC, p53, prealbumin (transthyretin), rhodopsin, S-antigen, and TGF-alpha. A single-base sequence polymorphism was identified in each of these gene sequences. Some of these polymorphisms were multiallelic and highly informative. Our results demonstrate the value of denaturing gradient gel electrophoresis for both identifying and analyzing human DNA polymorphisms. The ability to detect highly informative polymorphisms within gene sequences will greatly contribute to a gene-based polymorphic linkage map.     

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.     

Identification of polymorphisms by genomic denaturing gradient gel electrophoresis: application to the proximal region of human chromosome 21.

Genomic Denaturing Gradient Gel Electrophoresis (gDGGE) provides an alternative to the standard method of restriction fragment length polymorphism (RFLP) analysis for identifying polymorphic sequence variation in genomic DNA. For gDGGE, genomic DNA is cleaved by restriction enzymes, separated in a polyacrylamide gel containing a gradient of DNA denaturants, and then transferred by electroblotting to nylon membranes. Unlike other applications of DGGE, gDGGE is not limited by the size of the probe and does not require probe sequence information. gDGGE can be used in conjunction with any unique DNA probe. Here we use gDGGE with probes from the proximal region of the long arm of human chromosome 21 to identify polymorphic DNA sequence variation in this segment of the chromosome. Our screening panel consisted of DNA from nine individuals, which was cleaved with five restriction enzymes and submitted to electrophoresis in two denaturing gradient conditions. We detected at least one potential polymorphism for nine of eleven probes that were tested. Two polymorphisms, one at D21S4 and one at D21S90, were characterized in detail. Our study demonstrates that gDGGE is a fast and efficient method for identifying polymorphisms that are useful for genetic linkage analysis.     

DNA haplotype analysis suggests linkage disequilibrium in the human insulin receptor gene

Summary Haplotypes of the insulin receptor gene were resolved in parents from Scandinavian nuclear families by studying the segregation of seven restriction fragment length polymorphisms (RFLPs). Of 97 unrelated parents, 41 had non-insulin-dependent diabetes mellitus (NIDDM). Considerable linkage disequilibrium in the region of the insulin receptor gene was found. Pairwise non-random associations were found between proximate RFLP sites, indicating the absence of recombinational hot spots between these sites. Thus, association studies between DNA polymorphisms at this locus and disease susceptibility genes could well be feasible in this population. Differences in the distribution of insulin receptor haplotypes were examined between NIDDM patients and healthy subjects. However, the differences observed were not statistically significant.     

利用时间进程法优化活性污泥DG-DGGE图谱

目的:为了探讨电泳时间对双梯度-变性梯度凝胶电泳(DG-DGGE)分析活性污泥样品时的影响。方法:提取污泥DNA后,以通用引物338f/534r扩增16S rDNA序列,采用时间进程法优化PCR扩增产物的DG-DGGE分离效果。结果:采用不同电泳时间进行DGGE分析时,DGGE图谱存在显著的差异。16S rDNA V3区(200 bp)在凝胶梯度6%~12%,变性剂梯度30%~60%时,在200V电压下,最佳电泳时间为5h。     

Single base pair germ-line deletion in the p53 gene in a cancer predisposed family

A family with an aggregation of adrenocortical carcinoma, rhabdomyosarcoma, osteosarcoma, and early onset breast cancer was referred to our laboratory. Because this aggregation was reminiscent of Li-Fraumeni syndrome, germ-line mutation of the p53 tumor suppressor gene was sought in the DNA of two affected members. The highly conserved regions spanning exons 5 to 8 of the p53 gene were screened by a previously validated denaturing gradient gel electrophoresis method. A single base pair deletion at codon 215 was detected in constitutional DNA of the two patients, and in the DNA extracted from an adrenocortical carcinoma tumor specimen of the propositus. This deletion is predicted to lead to the formation of a truncated p53 protein, a relatively rare event in Li-Fraumeni families. The spectrum of tumors observed in this family does not differ markedly from the spectrum observed in families with missense p53 mutations.     

Cystic fibrosis in Lebanon: distribution of CFTR mutations among Arab communities

Cystic fibrosis (CF) is thought to be rare among the Arab populations from the Middle East and little data have been reported so far. We have studied a sample of 20 families living in Lebanon for several generations and who have at least one child with CF. These families are mainly from the Maronite, Greek Catholic, Greek Orthodox, Shiite or Sunnite groups. We found a 50% rate of consanguineous marriage, independent of the community of origin. The distribution of CF genotypes was determined through the screening of all exons of the CFTR (cystic fibrosis transmembrane conductance regulator) gene by the technique of denaturing gradient gel electrophoresis combined with asymmetric amplification DNA sequencing. A total of ten different mutations accounting for 87.5% of 32 unrelated CF alleles was identified, including two novel putative mutations (E672del and IVS21-28G→A). Three mutations, ΔF508 (37.5%), W1282X (15.6%), and N1303K (9.4%) accounted for 62.5% of CF alleles. Interestingly, in the Maronite group, 66.7% of the ΔF508 chromosomes were found to be associated with allele 7 of the IVS8(T)tract, contrasting with the absolute linkage disequilibrium between European ΔF508 chromosomes and allele 9. During this study, two previously undescribed polymorphisms (IVS14a + 17del5 and 2691T/C) were also identified. Received: 2 January 1997 / Accepted: 16 March 1997     

Detection of base mutations in genomic DNA using denaturing gradient gel electrophoresis (DGGE) followed by transfer and hybridization with gene-specific probes

It has been shown that minor differences, such as single-base-pair substitutions between otherwise identical DNA fragments can result in altered melting behavior detectable by denaturing gradient gel electrophoresis (DGGE). Sequence variations in only a small DNA region within one locus can be detected using the previously described procedures. We have developed a method for the efficient Southern transfer of genomic DNA fragments from the denaturing gradient gels in order to be able to analyze larger regions in several loci for variation. The gels were made using polyacrylamide containing 2% low-geling-temperature agarose (LGT). The polyacrylamide gel (PAG) was crosslinked with a reversible crosslinker, and after electrophoresis the crosslinks were cleaved, the structure of the gel being maintained by the agarose. After this treatment of the denaturing gels, more than 90% of the DNA fragments could be transferred to nylon membranes by alkaline transfer, while electroblotting transferred only 10% of the DNA. Hybridization with gene-specific probes was then performed. We have used this technique to identify an RFLP in the COL1A2 gene in a human genomic DNA sample. The transfer technique described should make the use of DGGE more widely applicable since the genomic DNA fragments separated on one gel can be screened with several different probes, both cDNA and genomic probes.     

Development of a polymerase chain reaction-based denaturing gradient gel electrophoresis technique to study nematode species biodiversity using the 18s rDNA gene

A polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) technique was developed to evaluate nematode biodiversity. Amplified fragments (approximately 630 bp) of nematode 18S rDNA could be separated by DGGE at 60 °C for 5.5 h using a gradient of 30–55% denaturant. No DNA from the nematodes’ food source was observed on the gels. The sensitivity of the system was tested using DNA from six species of Steinernema. The system clearly separated PCR fragments from all species except S. tami and S. carpocapsae, which had similar melting behaviour despite being widely separated on the phylogenetic tree.     

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.     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

Gene analysis of the N-terminal region of the estrogen receptor alpha in preeclampsia

Alterations in the NH(2)-terminal region of the estrogen receptor alpha (ERalpha) gene expressed in placental bed tissue may be implicated in the development of preeclampsia, the pathogenesis of which involves the spiral arteries. Therefore, mutations and polymorphisms on exons 1 and 2 of the gene encoding ERalpha were studied. Placental bed biopsies were taken from 20 healthy, normotensive pregnant women and 16 preeclamptic patients. DNA was extracted from the tissue and exon 1 and exon 2 were amplified by PCR prior to denaturing gradient gel electrophoresis analysis or to single stranded conformational polymorphism analysis. In exon 1, a codon 10 polymorphism, either homozygous for the wild type gene, homozygous for the mutant type gene, or heterozygous, was revealed in both patients and healthy individuals. A codon 87 polymorphism, homozygous for the wild type gene, was detected in both groups. No mutations or polymorphisms were found in exon 2. The allele distribution for either codon 10 or 87 between patients and healthy individuals showed no significant differences. In conclusion, genetic alterations in the NH(2)-terminal region of the ERalpha molecule are not correlated with preeclampsia.     

Majority of hMLH1 mutations responsible for hereditary nonpolyposis colorectal cancer cluster at the exonic region 15-16.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer susceptibility condition. Inherited mutations in at least four DNA mismatch repair genes, hMSH2, hMLH1, hPMS1, and hPMS2, are known to cause HNPCC. In this study we used denaturing gradient gel electrophoresis (DGGE) to screen for hMLH1 mutations in 34 unrelated HNPCC families (30 Dutch, 3 Italian, and 1 Danish). Ten novel pathogenic germ-line mutations (seven affecting splice sites, two frameshifts, and one in-frame deletion of a single amino acid) have been identified in 12 (35%) of these families. In a previous study, hMSH2 mutations were found in 21% of the same families. While the spectrum of mutations at the hMSH2 gene among HNPCC patients appears heterogeneous, a cluster of hMLH1 mutations has been found in the region encompassing exons 15 and 16, which accounts for 50% of all the independent hMLH1 mutations described to date and for > 20% of the unrelated HNPCC kindreds here analyzed. This unexpected finding has a great practical value in the clinical scenario of genetic services.     

Studies on the immunoglobulin V kappa locus in human lymphoid cell lines

DNA digests of 16 human lymphoid cell lines were studied in blot hybridization experiments with probes from V kappa genes and their immediate neighborhood as well as with single or low-copy probes from intergenic regions. The patterns were compared with those of placenta DNA digests in which the kappa genes are in the germline configuration. The differences of patterns which were detected with the first type of hybridization probes can be attributed to V kappa--J kappa rearrangements or to restriction site polymorphisms between individuals. Some of the pattern differences observed with the second type of probes can be interpreted best as arising from deletions of parts of the kappa locus. Such deletions may be individual variations but they may also be caused by the V kappa-J kappa rearrangement process. The results obtained with one particular probe which was derived from a nonduplicated part of the kappa locus allow some conclusions as to the mechanism of the V kappa--J kappa rearrangement: the genomic situation in some lymphoid cell lines can be explained by an inversion while in other cell lines clearly deletions have occurred. The observations are in agreement with the inversion-deletion mechanism of V kappa--J kappa rearrangement as proposed by Lewis et al. (1982, 1984).