共查询到20条相似文献,搜索用时 15 毫秒
1.
Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp 总被引:17,自引:0,他引:17
We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human beta-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes. 相似文献
2.
Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis 总被引:4,自引:0,他引:4
A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms
in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the
origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions
of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in
a human B-cell line TK6 and T-cells from four individuals. A T → C transition at position 16519 in the origin/membrane attachment
site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G → A transition
at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful
for the rapid screening of polymorphisms in a large number of samples.
Received: 19 October 1995 / Revised: 26 March 1996 相似文献
3.
We describe in detail a method which allows determination of the directions of replication fork movement through segments of DNA for which cloned probes are available. The method uses two-dimensional neutral-alkaline agarose gel electrophoresis followed by hybridization with short probe sequences. The nascent strands of replicating molecules form an arc separated from parental and nonreplicating strands. The closer a probe is to its replication origin or to the origin-proximal end of its restriction fragment, the shorter the nascent strands that are detected by the probe. The use of multiple probes allows determination of directions of replication fork movement, as well as locations of origins and termini. In this study, we used simian virus 40 as a model to demonstrate the feasibility of the method, and we discuss its applicability to other systems. 相似文献
4.
Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA-DRB3 alleles 总被引:4,自引:0,他引:4
B. M. Aldridge S. M. McGuirk R. J. Clark L. A. Knapp D. I. Watkins & D. P. Lunn 《Animal genetics》1998,29(5):389-394
The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3 -typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10–15% acrylamide combined with a 15–50% urea-formamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3 -typing. 相似文献
5.
G. E. Riedel S. L. Swanberg K. D. Kuranda K. Marquette P. LaPan P. Bledsoe A. Kennedy B.-Y. Lin 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1990,80(1):1-10
Summary We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms. 相似文献
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Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. 总被引:56,自引:8,他引:56 下载免费PDF全文
Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp. 相似文献
9.
Hybridization of DNA with oligonucleotides immobilized in a gel: a convenient method for recording single base replacements 总被引:2,自引:0,他引:2
K R Khrapko A A Khorlin I B Ivanov B K Chernov Iu P Lysov S K Vasilenko V L Florent'ev A D Mirzabekov 《Molekuliarnaia biologiia》1991,25(3):718-730
In an attempt to develop a reliable system for DNA sequence analysis with multiple hybridization probes, oligonucleotides down to 8 bases long were covalently immobilized in a thin layer of polyacrylamide gel fixed on a glass plate. It was shown possible to detect single base changes in DNA by hybridization of the immobilized oligonucleotides with radioactively and fluorescently labeled DNA fragments. Moreover, it was found that dissociation temperatures of differently GC-rich duplexes could be equalized by appropriate choice of immobilized oligonucleotides concentrations. A model accounting for this phenomenon is presented. In order to make the system more compact, a rectangular matrix of 200 mm dots of immobilized oligonucleotides ("hybridization chip") was designed which offered the sensitivity of 20 attomoles per dot for fluorescent DNA fragment. The applications and perspectives of the approach are discussed. 相似文献
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Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots. 下载免费PDF全文
M R Gray 《American journal of human genetics》1992,50(2):331-346
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. 相似文献
12.
W.W. Hauswirth P.J. Laipis M.E. Gilman T.W. OBrien G.S. Michaels M.A. Rayfield 《Gene》1980,8(2):193-209
Using a physical map of bovine mitochondrial DNA derived from the liver of a single Holstein cow, we have determined the location of the genes specifying the large and small riibosomal RNAs by hybridization analysis and electron microscopic observations of R-loop forms. Also, the position of the origin of DNA replication (D-loop) has been located by electron microscopy. Additionally, the direction of D-loop expansion and the polarity of the large and small ribosomal RNA genes were determined. 相似文献
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Farnleitner AH Kreuzinger N Kavka GG Grillenberger S Rath J Mach RL 《Letters in applied microbiology》2000,30(6):427-431
A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types. 相似文献
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The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method. 相似文献
16.
J S Fawcett D Wheeler A Chrambach 《Journal of biochemical and biophysical methods》1992,24(3-4):181-194
Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180). 相似文献
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Detection of a single base exchange in PCR-amplified DNA fragments using agarose gel electrophoresis containing bisbenzimide-PEG. 下载免费PDF全文
Using PCR fragments of known sequences derived from isolates of two related fungal species, simple submarine electrophoresis in agarose gels containing a bisbenzimide-PEG conjugate (H.A.-Yellow) has been shown to be capable of distinguishing DNA fragments 567 bp long which differ by as little as a single base change. However, only changes affecting bisbenzimide binding sites (which consist of at least four consecutive A/T bases) alter mobility; other changes are ineffective. A second ligand (H.A.-Red) with high G/C specificity is suggested which may be as effective in detecting other sequence changes. 相似文献
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Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases 总被引:1,自引:0,他引:1
《Cell》2022,185(10):1764-1776.e12
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