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.     

A rapid and efficient procedure for the purification of DNA from agarose gels

DNA fragments electrophoresed through a horizontal agarose slab gel can be recovered by inserting strips of filter paper backed by dialysis membrane into slits cut in the gel in front of the DNA bands and continuing electrophoresis until the DNA is collected in the paper. Elution of the DNA from the filter paper is then achieved by low-speed centrifugation. Recovery well above 70% is routinely obtained with this technique and the DNA recovered is biologically active and can be recleaved, ligated, labeled in vitro by nick translation and hybridized to RNA.     

Transfer of small DNA fragments from polyacrylamide gels to diazobenzyloxymethyl-paper and detection by hybridization with DNA probes.

A method for transferring small DNA fragments from composite polyacrylamide-agarose gels to diazobenzyloxymethyl (DBM)-paper is described. DNA fragments are separated by electrophoresis in polyacrylamide-agarose gels crosslinked with N,N′-diallyltartardiamide instead of N,N′-methylenebis-acrylamide. The crosslinks are cleaved by treating the gel with periodic acid after electrophoresis and the DNA is denatured with alkali. After neutralization, the single stranded DNA fragments are transferred to DBM-paper and detected by hybridization with labeled DNA probes. The procedure has been used to transfer and visualize unlabeled SV40 DNA fragments in the size range 28 to 1790 base pairs.     

Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I

Circular (e.g. simian virus 40) and linear (e.g. λ phage) DNAs have been labeled to high specific radioactivities (>10⁸ cts/min per μg) in vitro using deoxynucleoside [α-³²P]triphosphates (100 to 250 Ci/mmol) as substrates and the nick translation activity of Escherichia coli DNA polymerase I. The reaction product yields single-stranded fragments about 400 nucleotides long following denaturation. Because restriction fragments derived from different regions of the nick-translated DNA have nearly the same specific radioactivity (cts/min per 10[su3] bases), we infer that nicks are introduced, and nick translation is initiated, with equal probability within all internal regions of the DNA. Such labeled DNAs (and restriction endonuclease fragments derived from them) are useful probes for detecting rare homologous sequences by in situ hybridization and reassociation kinetic analysis.     

Rapid alkaline transfer of low molecular weight DNA from NuSieve GTG agarose gels

The rapid alkaline transfer of high molecular weight DNA from agarose gels to nylon membranes has greatly decreased the time required for setup of Southern transfers. This technique has been used to resolve genomic DNA greater than 1000 base pairs by conventional electrophoresis on 1% agarose gels followed by alkaline transfer to nylon membrane. Now we report that this rapid alkaline method can be used for the transfer of low molecular weight DNA fragments (10 to 1000 base pairs) from NuSieve GTG agarose gels to nylon membrane.     

Southern blotting

This protocol describes a basic method to perform the Southern blot. Blotting allows the detection of specific molecules among a mixture separated by gel electrophoresis. Molecules are transferred from the gel to a porous membrane by capillary action using absorbent paper to soak solution through the gel and the membrane. For DNA, specific sequences are detected in the membrane by molecular hybridization with labeled nucleic acid probes. The original method, on which this protocol is based, used labeled RNAs to detect specific DNA fragments in genomic DNA that had been digested with restriction endonucleases. This protocol can be completed in 1-5 d and is inexpensive to carry out, as it requires only basic laboratory equipment.     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

Determination of the relative positions of amino acids by partial specific cleavages of end-labeled proteins

We have developed a new method for obtaining information about protein sequences that uses an approach analogous to that used to determine DNA sequences. In essence, three steps are involved. First, a detectable label is attached exclusively to the amino terminus of a polypeptide. Next, the labeled chain is subjected to partial specific cleavage in a way that produces roughly equimolar amounts of fragments of different sizes. Cleavages for methionine, tryptophan, arginine, aspartyl-proline bonds, and asparaginyl-glycine bonds have been employed. Lastly, the labeled fragments are separated according to size by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The distribution of target amino acids along the polypeptide chain can be deduced from the specific pattern of labeled bands by reading the "ladder" in the same way that DNA sequencing gels are read. Although the method can be conducted with a radioactive label, we have chosen to use a fluorescent label. We have applied the method successfully to the three subunit chains of two different fibrinogens.     

~(35)S标记探针检测人类单拷贝基因

应用改进的缺口翻译方法,将α-~(35)S-dATP参入DNA分子,获得了>10~8dpm/μgDNA的高比度~(35)S标记探针。用其做Southern吸印杂交探测人类单拷贝基因组织,放射自显影4～10天,能够得到满意的力谱。与~(32)P相比,~(35)S具有半衰期长,无辐射伤害,价格便宜等优点。     

Nick translation of HeLa cell nuclei as a probe for locating DNase I-sensitive nucleosomes

The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.     

A cloned chromosomal DNA fragment which differentiates Yersinia pestis from Yersinia pseudotuberculosis and Yersinia enterocolitica

Chromosomal DNA from reference Yersinia strains was digested individually with 9 restriction endonucleases. DNA fragments were separated and analyzed by electrophoresis through agarose gels. The clearest fragment patterns were obtained when EcoRI was employed. The Y. pestis fragment pattern obtained after the use of this enzyme showed the presence of a unique DNA fragment with molecular mass 1400 bp. This DNA fragment was cloned, purified, labeled with 32P and then used to probe EcoRI digests of all three Yersinia species. A strong hybridization signal was obtained with Y. pestis strain. No such signal was found with Y. pseudotuberculosis or Y. enterocolitica. These results indicate that the DNA fragment is species specific and could be used as a diagnostic DNA probe for Y. pestis.     

Semi-dry electroblotting of DNA

A procedure is described for the rapid transfer of DNA from agarose gels to nylon membranes using the semi-dry electroblotting technique. A Hind III digest of lambda DNA which was separated in a 1% agarose gel containing Tris, Borate, and EDTA (pH 8.0) was employed for the electrotransfer experiments. Transfer efficiency was determined by staining the DNA on the nylon membranes with a colloidal iron reagent. Current densities of 3-5 mA/sq. cm of gel permitted the transfer of high (23 kb) and low (0.3 kb) molecular weight fragments within 15 min. However, efficient transfer required a high ionic strength buffer that would prevent uneven dehydration of the agarose gel. Critical parameters for the transfer of nucleic acids with the semi-dry technique are discussed.     

'Northern Cross' hybridization for rapid identification of exon-containing restriction fragments

The Northern Cross method allows direct comparison of restriction digests of cDNA and genomic clones to RNA populations by a specialized form of hybridization. This technique is based on the use of Northern and Southern blotting techniques and requires the use of two nylon membranes of differing chemical characteristics. A nylon membrane containing permanently affixed, electrophoretically fractionated RNAs is contact-hybridized at a right angle to a second, chemically different nylon membrane containing transiently bound, fractionated labeled DNA fragments. RNA and DNA bands possessing homology will hybridize where they cross, forming an autoradiographically detectable spot. This Northern Cross procedure proportionately represents the amounts of different RNAs derived from a particular sequence in a manner similar to what would have been observed in a Northern blot. This method, which can be used in the analysis of even relatively rare RNA species, permits rapid and fairly inexpensive identification of exon-containing fragments or determination of the relationship between related, multiple RNA species.     

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.     

DNA chip replication for a personalized DNA chip

We report the replication technology of DNA chip using by sequence specific localization of nucleic acids via hybridization and electric transfer of the nucleic acids onto a new substrate without losing their array information. The denatured DNA fragments are first spotted and UV-cross-linked on a nylon membrane. The membrane is then immersed and hybridized in a DNA mixture solution that contains all complementary sequences of the nucleic acids to be hybridized with the DNA fragments on the membrane. The hybridized DNA fragments are transferred to another membrane at the denatured condition. After separating two membranes, the transferred membrane contains a complementary array of DNA fragments. This method can be used for the replication of the same copy of DNA chip repeatedly and moreover could be applied for a personalized DNA chip fabrication, where specific information of each spot of DNA chip is originated from the genetic information of a personal sample.     

Two methods to detect DNA fragments produced by restriction enzymes

This report summarizes two methods for detecting limited amounts of DNA from restriction endonuclease digests. The first is a photographic system for visualizing ethidium bromide-stained DNA fragments in agarose gels which can detect as little as 50-100 pg DNA per band. The second technique is direct sulfonation of DNA fragments bound to nylon membranes followed by visualization of the fragments by nonradioactive immunoblot methods. The immunohistochemical staining can detect 10 pg DNA per band. The direct sulfonation technique is not intended to identify specific DNA sequences; DNA-DNA hybridization with sulfonated probes has previously been described (P. Lebacq, D. Squalli, M. Duchenne, P. Poulety, and M. Johannes (1988) J. Biochem. Biophys. Methods 15, 255-266). Direct sulfonation can be used when samples are relatively free of contaminating nucleic acids and is a useful alternative to end-labeling. These highly sensitive techniques may be suitable when the DNA source is of limited quantity or in instances where radiolabeling is not permitted.     

Internal fluorescence labeling with fluorescent deoxynucleotides in two-label peak-height encoded DNA sequencing by capillary electrophoresis.

Fluorescently labeled deoxynucleotides were used for internal labeling of DNA sequencing fragments generated in a two-color peak-height encoded protocol. Sequenase and a manganese-containing buffer were used to generate uniform peak heights. Tetramethyl rhodamine - dATP was used in a labeling step, followed by termination with ddATP and ddCTP in a 3:1 ratio. Fluorescein - dATP was used in a second reaction, followed by termination with ddGTP and ddTTP in a 3:1 ratio. The fragments were pooled and separated by capillary gel electrophoresis. The results were compared with peak-height encoded sequencing based on fluorescently labeled primers. The dye-labeled primers produced higher resolution separations for shorter fragments. However, dye-labeled primer fragments suffered from an earlier onset of biased reptation and produced shorter sequencing reads. Fragments from 50 to at least 500 bases in length could be sequenced with the internal labels.