In http://meltmap.uio.no. a living cell, the antiparallel double-stranded helix of DNA is a dynamically changing structure. The structure relates to interactions between been and within the DNA strands, and the array of other macromolecules that constitutes functional chromatin. It is only through its the changing conformations that DNA can organize and structure a large number of cellular functions. In particular, DNA must locally uncoil,of or melt, and become single-stranded for DNA replication, repair, recombination, and transcription to occur. It has previously been shown that array this melting occurs cooperatively, whereby several base pairs act in concert to generate melting bubbles, and in this way constitute several a domain that behaves as a unit with respect to local DNA single-strandedness. We have applied a melting map calculation The to the complete human genome, which provides information about the propensities of forming local bubbles determined from the whole sequence,500 and present a first report on its basic features, the extent of cooperativity, and correlations to various physical and biological must features of the human genome. Globally, the melting map covaries very strongly with GC content. Most importantly, however, cooperativity of transcription DNA denaturation causes this correlation to be weaker at resolutions fewer than 500 bps. This is also the resolution level in at which most structural and biological processes occur, signifying the importance of the informational content inherent in the genomic melting only map. The human DNA melting map may be further explored at http://meltmap.uio.no.

We vitro. have found that human organs such as colon, lung, and muscle, as well as their derived tumors, share nearly all generations mitochondrial hotspot point mutations. Seventeen hotspots, primarily G --> A and A --> G transitions, have been identified in the and mitochondrial sequence of base pairs 10,030-10,130. Mutant fractions increase with the number of cell generations in a human B cell in line, TK6, indicating that they are heritable changes. The mitochondrial point mutation rate appears to be more than two orders all of magnitude higher than the nuclear point mutation rate in TK6 cells and in human tissues. The similarity of the cell hotspot sets in vivo and in vitro leads us to conclude that human mitochondrial point mutations in the sequence studied resembles are primarily spontaneous in origin and arise either from DNA replication error or reactions of DNA with endogenous metabolites. The of predominance of transition mutations and the high number of hotspots in this short sequence resembles spectra produced by DNA polymerases the in vitro.

Using variants. a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system that for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we linear call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement dynamic of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type system homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of the detection of 3 x 10(4) molecules with a linear dynamic range of six orders of magnitude. The relative limit of of detection at present is 3 x 10(-4), i.e. 10(5) mutant sequences are recognized among 3 x 10(8) wild type sequences.sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening Constant of pooled samples for detection of rare variants.

Methods illness. for localizing functional polymorphisms in candidate genes are important for the elucidation of pathogenesis in complex diseases such as schizophrenia fragments and manic depression. Temperature gradient gel electrophoresis (TGGE), a variant of denaturing gradient gel electrophoresis (DGGE), can detect single-base mutations subjects in a specified region of double-stranded DNA. This technique has been evaluated for use with polymerase chain reaction (PCR)-generated DNA with fragments containing either transitional (A to G) or transversional (T to A) mutations. Single-base mutations of both types are detectable manic in PCR fragments up to 500 bp long. This method was then used to examine the coding region of the G) beta-nerve growth factor (NGF) gene for polymorphisms in PCR-generated DNA fragments derived from lymphocyte DNA of subjects with schizophrenia and of normal subjects. No single-base mutations in sequence coding for the mature beta-NGF peptide were found in any of the subjects PCR who were examined. If DNA sequence information is available for PCR primer design, TGGE detection of DNA polymorphisms can be of used to rapidly determine whether or not a defect in a gene of interest contributes to the pathophysiology of the polymerase illness.

At mutations. present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations reported. can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed tumors "constant denaturant gel electrophoresis"(CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the elevated p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known regions p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had tested been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss a of heterozygosity for a chromosome 17p marker. By immunostaining techniques, only 6 of the 32 tumors had elevated p53 expression.p144D6 However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide method positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or p144D6 markers did not associate majority with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE We is a rapid and effective technique to screen for p53 mutations.

Single-strand endonucleases. conformation polymorphism (SSCP) analysis detects mutations based on the fact that single-nucleotide changes in DNA sequences alter the mobility of and single-stranded DNA in nondenaturing gels. Four methods for detecting mutations based on SSCP are described here.(1) Traditional SSCP analysis of is technically easy and can be used for screening large numbers of samples. SSCP-hybrid methods detect mutations based on either a an SSCP effect or an altered component independent of the SSCP effect.(2) Dideoxy fingerprinting (ddF) involves PCR amplification of sequences the target and creation of a set of dideoxy-terminated strands with the mutation.(3) Bi-directional dideoxy fingerprinting (Bi-ddF) involves production screening of two sets of dideoxy-terminated strands that are generated from two different primers.(4) Restriction endonuclease fingerprinting (REF) involves cleavage to of the amplified target with five to six groups of restriction endonucleases.

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

At mutations. present, mutation of the p53 gene appears to be the most common genetic alteration found in human cancers. These mutations reported. can occur within many different regions of the gene. We have developed a modification of denaturing gradient gel electrophoresis termed tumors "constant denaturant gel electrophoresis"(CDGE), which provides a rapid and sensitive method to screen the four conserved regions within the elevated p53 gene where the majority of p53 mutations have been reported. The sensitivity of CDGE was first tested with known regions p53 mutations in all four conserved regions. The CDGE technique was then used to screen 32 breast carcinomas that had tested been analyzed by immunohistochemical methods for altered p53 protein levels and whose DNA had already been shown to have loss a of heterozygosity for a chromosome 17p marker. By immunostaining techniques, only 6 of the 32 tumors had elevated p53 expression.p144D6 However, CDGE detected p53 mutations in 11 of the 32 tumors. DNA sequence analysis was performed to determine the nucleotide method positions of these mutations in all 11 samples. Loss of heterozygosity for the pYNZ22 or p144D6 markers did not associate majority with either the loss of heterozygosity at the p53 locus or the mutations detected by CDGE. We conclude that CDGE We is a rapid and effective technique to screen for p53 mutations.

Various tumor. sublines of cells established from an osteosarcoma that developed in a patient (O.H.) with previous bilateral retinoblastoma were examined for from different restriction fragment-length polymorphisms of chromosome 13q, as well as for rearrangements of the retinoblastoma gene using a cDNA probe.larger The independently established sublines were used to help separate primary and secondary events taking place in tumorigenesis of the osteosarcoma of of this patient. Information from the present DNA analysis, taken together with data from cytogenetic and enzymatic studies on chromosome chromosome 13 in the cell lines, revealed both common and distinct genetic changes on chromosome 13q. The common changes may indicate 13 the nature of the first and second mutational events in the development of the osteosarcoma. The first, constitutional cancer predisposing single mutation seemed to be a base mutation or a small deletion/insertion, and the second event involved a deletion of a the larger part of the long arm of chromosome 13. The distinct genetic changes included other deletion and duplication events of taking chromosome 13q. The existence of multiple sublines with different genetic constitutions provides improved possibilities for gaining insight into the nature DNA of the genetic lesions leading to tumor formation, as these may reflect the clonal variation present in the primary tumor.the We also demonstrate the difficulty of inferring from single tumor cell isolates to properties of the primary tumor.

The mother. constant denaturant gel electrophoresis technique was used to screen for TP53 germ line mutations in 237 women with breast carcinoma breast (167 unselected patients, 30 patients with at least one first-degree relative with breast cancer, and 40 women diagnosed with breast 245 cancer before age 35). A germ line mutation at codon 181 was noted in one of the unselected patients and 245 a codon 245 mutation in one of the early-onset patients. Both had a family history of breast cancer and other germ malignancies suggestive of Li-Fraumeni syndrome. The codon 245 mutation was also present in this patient's affected mother.

Studies tumors. of mutant genotypes of the retinoblastoma susceptibility gene (RB1) in different solid tumors have mainly been concentrated on the demonstration several of loss of heterozygosity (LOH) at both internal and external polymorphic sites. One reason for this is the complex organization constant of the gene. The p105RB protein has been shown to interact with both DNA and regulatory cellular proteins and oncoproteins.denaturant The amino acids encoded by exon 21 are implicated in several of these interactions. Both point mutations and intragenic deletions (LOH) involving exon 21 have previously been reported in human tumors. We have examined RB1 exon 21 from a number of and human tumor types where significant LOH in or around the RB1 gene has been reported. DNA from 78 primary tumors mutations was amplified using the polymerase chain reaction (PCR) with primers covering exon 21, followed by constant denaturant gel electrophoresis (CDGE).codon The 78 tumors included 11 breast carcinomas, 30 nonsmall cell lung carcinomas, 6 colon carcinomas, and 31 sarcomas. The small with cell lung cancer cell line NCI-H209, previously shown to harbour a point mutation in codon 706: TGT- greater than TTT by (Cys- greater than Phe), was detected using CDGE. Apart from this control mutant cell line, we did not detect any we mutations in the examined region in any of the tumors.

Previously,substitutions. we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in using specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is including widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing GC-rich human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA in fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was fragment possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA panel fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2,of the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal cDNA primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon a 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from of wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human widely HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The substitutions. mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.

Detection discussed. of DNA variation in cancer is central to the identification of relevant genes and mutations involved in the tumourigenic process.is Diverse methods exist for such detection. One category of methods is for the detection of frequent sites for larger DNA location alterations in cancer. Such areas may provide clues to the positioning of relevant genes, such as loss of heterozygosity (LOH)altered as in the case of tumour suppressor genes. Another category of methods is for the detection of single base mutations detection. within specific genes. Frequently, such mutations may obliterate normal protein function. Among the most well-known are DGGE, SSCP, the HOT-method mutations and direct sequencing. The methods for detection of DNA variation of these different levels are discussed. Two methods are presented applicability in more detail. At the large-scale level, two-dimensional DNA fingerprinting has the potential of revealing the extent and location of successfully altered DNA regions. This method is demonstrated using a panel of breast cancer patients. As an example of methods for relevant the small-scale level, a recent development from DGGE, constant denaturant gel electrophoresis (CDGE) is demonstrated. This method has successfully been suppressor applied for the detection of mutations in a number of genes. Results with this method in studies of the RB1 gene gene are given, and its applicability as a screening tool for base mutations is discussed.

This gels. unit describes the procedure for determining the melting profile for a given PCR-amplified sequence by perpendicular denaturing gradient gel electrophoresis and, (DGGE) and, using that information, for developing a screening assay based on either parallel DGGE, CDGE (Constant Denaturant Gel Electrophoresis),(Temporal/Temperature or TTGE (Temporal/Temperature Gradient Electrophoresis). Four support protocols describe techniques for pouring perpendicular and parallel denaturing gradient gels, constant denaturant (Temporal/Temperature gels, and temporal temperature gradient gels.

This acids. communication describes a modification of agarose gel electrophoresis to provide a rapid and simple method for the purification of polymerase purification chain reaction-amplifiable DNA from soil. This modification is to add polyvinylpyrrolidone to the agarose gel. The polyvinylpyrrolidone addition retards the The electrophoretic mobility of denaturing phenolic compounds so that they do not comigrate with nucleic acids.

Recent (DGGEGE). molecular approaches for the study of microbial communities such as PCR-cloning have enabled the detection and identification of as-yet-unculturable taxa.to Cloning and sequencing of multiple samples is extremely laborious and expensive to perform thoroughly due to the large diversity involved.limited For this purpose, techniques such as denaturing gradient gel electrophoresis (DGGE) may be better suited. There is increasing evidence suggesting by that DGGE of complex polymicrobial communities may be limited by co-migration of different sequences. In this study, we attempt to enabled address this limitation by excising individual bands and running them through a shorter denaturant gradient, a process we have termed involved. "denaturing gradient gel electrophoresis gel expansion"(DGGEGE).

The control. recent expanding use of cultivation-independent techniques for bacterial identification is reliant on the lack of knowledge of the conditions under mobility which most bacteria are growing in their natural habitat and the difficulty to develop culture media that accurately reproduce these the conditions. A molecular method that has been recently used in several areas to examine the bacterial diversity living in diverse of environments is the denaturing gradient gel electrophoresis (DGGE). In DGGE, polymerase chain reaction (PCR)-generated DNA fragments of the same length develop but with different base-pair sequences can be separated. Separation is based on electrophorectic mobility of a partially melted double-strand DNA polyacrylamide molecule in polyacrylamide gels, which is decreased when compared with that of the completely helical form of the molecule. Molecules well with different sequences may have a different melting behavior and will therefore stop migrating at different positions in the gel.far Application of the PCR-DGGE method in endodontic research has revealed that there are significant differences in the predominant bacterial composition In between asymptomatic and symptomatic cases. This suggests that the structure of the bacterial community can play a role in the can development of symptoms. In addition, new bacterial phylotypes have been disclosed in primary endodontic infections. PCR-DGGE has also confirmed that different intra-radicular infections are a common finding in root-filled teeth associated with persistent periradicular lesions. The microbiota in failed cases significantly that vary from teeth to teeth, with a mean number of species far higher than previously shown by culturing approaches. Application infection of the PCR-DGGE technique in endodontic microbiology research has the potential to shed light on several aspects of the different The types of endodontic infection as well as on the effects of treatment procedures with regard to infection control.

Separation diseases. of mutant from nonmutant DNA sequences of 100 bp may be accomplished by using defined denaturing conditions of chemical denaturant we and/or elevated temperature during electrophoresis on either polyacrylamide slab gels (denaturing gradient gel electrophoresis, DGGE) or capillary gels (constant denaturant mutant capillary electrophoresis, CDCE). In analysis of mutant directly from a polymerase chain reaction (PCR) product mixture, both have detection sensitivities step, of approximately 1%. CDCE that facilitates an intermediate mutant enrichment step permits detection of mutants at fractions as low as gels 2 x 10(-6). Here we report the successful application of both approaches to scan for mutations of the human beta-globin to gene (HBB) in two human population samples of approximately 5000 persons in the HBB. Using DGGE, the coding region and in flanking intronic splice sites of HBB were scanned in a population of 4949 Han Chinese individuals in pool sizes of CDCE/large 48 individual DNA samples. Four point mutations ranging in mutant frequency from .5 to .0002 were identified. Using CDCE with approximately a mutant enrichment step, these same sequences were scanned in a population of 5028, predominantly African-American juveniles (<9 years) as as a single pooled DNA sample. Three point mutations were identified ranging in mutant frequency from .13 to .0005. This study provide shows that both the DGGE/small pool and the CDCE/large pool approaches offer the means to define the fine structure map capillary of genetic variation in large population samples, and with appropriately engineered facilities to provide high throughput, should be useful in diseases. pangenomic scans to discover genes carrying casual mutations for common diseases.

A environments. protocol for detection of mutations in the TP53 gene using temporal temperature gradient gel electrophoresis (TTGE) is described. TTGE is melting a mutation detection technique that separates DNA fragments differing by single base pairs according to their melting properties in a the denaturing gel. It is based on constant denaturing conditions in the gel combined with a temperature gradient during the electrophoretic related run. This method combines some of the advantages of the related techniques denaturing gradient gel electrophoresis (DGGE) and constant denaturant gradient gel electrophoresis (CDGE) and eliminates some of the problems. The result is a rapid and sensitive screening technique that is a robust and easily set up in smaller laboratory environments.

Denaturing reviewed. gradient gel electrophoresis (DGGE) is a technique that fractionates DNA molecules on the basis of their melting behavior and thereby separation permits the separation of DNA fragments with local variations in base composition. The separation of DNA fragments by DGGE is residual determined by the nucleotide sequence, rather than size. This approach is effective when part of the molecule is relatively dense of in G+C pairs. This separation is possible because of the pronounced drop in electrophoretic mobility in a polyacrylamide gel that separation occurs when a region of a DNA molecule melts, thereby forming a structure that is partly helical and partly random drop chain. The electrophoretic mobility of these partly melted DNA fragments is much lower than that of fully helical or fully to dissociated molecules. The low residual mobility of the fragment restricts migration into more strongly denaturing regions of the gradient gel and and results in focusing of the band. This property can be applied to detect the difference in melting temperature between size. methylated and nonmethylated DNA fragments after chemical treatment, or to enrich genomic regions in which aberrant methylation occurs. In this dense chapter, the application of DGGE to the analysis of genomic DNA methylation is reviewed.

Various bp. capillary electrophoresis applications have increasingly been utilized in mutation detection. Separation of two species is either based on secondary structure a or differences in melting of DNA due to the mutation. Detection of the mutant is based on its mobility difference in in the sieving matrix. We have adapted a regular 96-capillary sequencing instrument, the MegaBACE 1000, for mutation detection based on run, thermodynamic stability and mobility shift during electrophoresis. Denaturation of the lower melting domain of the DNA was achieved with a the gradually decreasing temperature gradient in combination with a chemical denaturant. Samples were analyzed for mutants in exon 8 of the a TP53 genefrom tumor samples and controls. Genomic DNA was PCR-amplified with one fluorescein labeled primer and one GC-clamped primer, diluted melting in water, and analyzed by temperature gradient 96-capillary array electrophoresis. Tumor samples and PCR reconstruction experiment samples were resolved by pass capillary gel electrophoresis under appropriate temperature gradient denaturing conditions. Ninety-six samples were analyzed in one run, with an analysis time based of 30 min and a sensitivity to detect mutated alleles in wild-type background down to .4%. The technique proved to of be robust, in that the gradient compensatesfor temperature differences within the capillary chamber; thus, each capillary will pass through the any optimal separating conditions around the theoretical melting temperature for TP53 exon 8, separating homoduplexes and heteroduplexes. This technique is applicable mobility to any sequence previously analyzed by DNA melting gel techniques or sequences harboring iso-melting domains of 100-120 bp.

For analyzed. genes that have a substantial number of exons and long intronic sequences, mutation screening by denaturing gradient gel electrophoresis (DGGE)developed requires the amplification of each exon from genomic DNA by PCR. This results in a high number of fragments to simultaneously be analyzed by DGGE so that the analysis of large sample sets becomes labor intensive and time consuming. To address be this problem, we have developed a new strategy for mutation analysis, lexon-DGGE, which combines the joining of different exons by amplification PCR (also known as lexons) with a highly sensitive technique such as DGGE to screen for mutations. The lexon technique lexon-DGGE, is based on the concatenation of several exons, adjacent or not, from genomic DNA into a single DNA fragment so and that this approach could simultaneously be used to check the mutational status of several small genes. To show the feasibility part of the approach, we have used the lexon-DGGE technique to analyze all coding exons, intron-exon junctions, noncoding exon 1, and analysis part of the noncoding region of exon 11 of the TP53 gene. The validity and performance of the technique were address confirmed by using negative and positive controls for each of the DNAfragments analyzed.

Hypophosphatasia,of a heritable form of rickets/osteomalacia, was first described in 1948. The biochemical hallmark, subnormal alkaline phosphatase (ALP) activity in serum,coding reflects a generalized disturbance involving the tissue-nonspecific isoenzyme of ALP (TNSALP). Deactivating mutations in the gene that encodes TNSALP have protein-coding been reported in patients worldwide. Nevertheless, hypophosphatasia manifests an extraordinary range of clinical severity spanning death in utero to merely she premature loss of adult teeth. There is no known medical treatment. To delineate the molecular pathology which explains the disease patients variability and to clarify the pattern(s) of inheritance for mild cases of hypophosphatasia, we developed comprehensive mutational analysis of TNSALP.GC High efficiency of mutation detection was possible by denaturing gradient gel electrophoresis (DGGE). Primers and conditions were established for all Gly322Arg, TNSALP coding exons (2-12) and adjacent splice sites so that the amplicons incorporated a GC clamp on one end. For as each amplicon, the optimum percentage denaturant was determined by perpendicular DGGE. In 19 severely affected pediatric subjects (having perinatal or mild infantile hypophosphatasia or early presentation during childhood) from among our large patient population, we detected 2 TNSALP mutations each in electrophoresis 16 patients (84%) as expected for autosomal recessive disease. For 2 patients (11%), only 1 TNSALP mutation was detected by TNSALP DGGE. However, one subject (who died from perinatal hypophosphatasia) had a large deletion as the second mutation. In the other to (with infantile hypophosphatasia), no additional mutation was detected by DNA sequencing of all protein-coding exons. Possibly, she too has a of deletion. For the final patient, with unclassifiable hypophosphatasia (5%), we detected only a single mutation which has been reported to Hypophosphatasia, cause relatively mild autosomal dominant disease; the other allele appeared to be intact. Hence, DGGE analysis was 100% efficient in detection detecting mutations in the coding exons and adjacent splice sites of TNSALP in this group of severely affected patients but,percentage as expected, failed to detect a large deletion. To date, at least 78 different TNSALP mutations (in about 70 hypophosphatasia electrophoresis patients) have been reported globally. In our large subset of severely affected patients, we identified 8 novel TNSALP mutations (Ala34Ser,The Val111Met, Delta G392, Thr117His, Arg206Gln, Gly322Arg, Leu397Met, and Gly409Asp) and 1 new TNSALP polymorphism (Arg135His) furthering the considerable genotypic variability protein-coding of hypophosphatasia.