Identification of the aph IV gene from protoplast-derived maize plants by a simple nonradioactive DNA hybridization method

Summary Protoplast-derived, transformed maize plants were evaluated by Southern analysis for the presence of the aph IV gene which codes for resistance to the antibiotic, hygromycin B. This gene was used as a selectable marker for the transformation of maize protoplasts. Southern analysis was performed with fluorescein-labeled probe DNA. A new method for labeling molecular weight markers with fluorescein-N⁶ is presented. The nonradioactive Southern analysis method is compared to the radioactive method and the results show that the nonradioactive method is as sensitive as the radioactive method.     

A simple method for detecting protein antigens in flour.

Wheat, barley, rye or oat flour was dissolved in 0.2N NaOH using magnetic stirring followed by sonication. The ensuing clear solutions contained 95% of protein in the dry matter. Aliquots were electrophoresed on 12% polyacrylamide gels which were either stained with Coomassie brilliant blue or Western-blotted on nitrocellulose membrane. Incubation of the latter with serum from persons sensitized to flours allowed the detection of antigenic flour protein classes. It seems that many antigens are present in common kitchen flour.     

A simple method for detecting drug effects on the DNA of mammalian cells

A simple method for detecting drug or X-ray effects on the DNA of intact cells is described. This method uses an easily constructed viscometer to measure changes in the viscosity of alkaline cell lysates. The method has been tested by comparing the effects of 4′-[(9-acridinyl)amino]methanesulfon-o-anisidide (oAMSA), 4′-[(9-acridinyl)amino]methanesulfon-m-anisidide (mAMSA), daunorubicin, and adriamycin on cellular DNA.     

A simple method for detecting heparinase-producing bacteria

A simple method for detecting heparinase-producing bacteria is described. The method is based on the metachromatic reaction between heparin and toluidine blue. Though developed primarily for Bacteroides spp., the method should find application in the detection of other aerobic and anaerobic heparinase-producing bacteria, without the need for large amounts of media and expensive spectrophotometric equipment.     

A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.

We have developed a novel nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins. This method is derived from previously described protocols developed for the same purpose by using radioactively labeled DNA probes containing protein recognition sequences. This nonradioactive strategy relies upon the use of a small hapten, digoxigenin. Fusion proteins expressed from the recombinant bacteriophage lambda gt11/lambda ZAP are immobilized on nitrocellulose filters and probed with digoxigenin-labeled double-stranded DNA as a ligand. The specifically bound DNA probes can be detected through sequential incubations with antibody-enzyme conjugate and enzyme substrates. This technique has been successfully utilized to isolate several cDNA clones encoding DNA binding proteins.     

A nonradioactive method for measuring DNA damage and its repair in nonproliferating tissues

The fluorometric assay of Kissane and Robins has been modified to monitor DNA in alkaline sucrose gradient fractions. Using this procedure the sedimentation analysis of DNA not only of liver, but also of brain, thymus, lung, pancreas, kidney, and skin was carried out. Like liver DNA, DNA released by the alkaline lysis of the above organs sedimented as heavy DNA (> 1 × 10⁹ daltons). A good correspondence was obtained for the sedimentation profiles of liver DNA whether DNA in the gradient fractions was determined by the fluorometric method or by measuring radioactivity.Using the fluorometric assay, the strand breaks not only of liver DNA but also of brain and kidney DNA have been demonstrated following the intravenous administration ofN-methylnitrosourea. Carcinogen-induced DNA damage and repair (as measured by sedimentation of DNA in alkaline sucrose gradients) in any organ including human biopsy specimens are potentially measurable by this procedure.     

A simple method for DNA restriction site mapping.

When a DNA molecule, enzymatically labelled with 32p at one end, is partially digested with a restriction enzyme labelled tdna fragments are obtained which form an overlapping series of molecules, all with a common labelled terminus. ta restriction map can then be constructed from an analysis of the size distribution of these molecules. This technique has been used for the restriction site mapping of cloned histone DNA (h22) where as many as 35 cleavage sites may be accurately determined in a single experiment.     

A simple and sensitive method for detecting bacterial elastase production

A sensitive method for detecting bacterial elastase production in growing cultures is described. A variety of commonly isolated clinically relevant aerobic and anaerobic bacteria have been shown to produce the enzyme.     

An alternative nonradioactive method for labeling DNA using biotin

An alternative nonradioactive labeling method and a highly sensitive technique for detecting specific DNA sequences are described. The labeling method requires the "Klenow" fragment of DNA polymerase I and random hexanucleotides (synthesized or naturally extracted) as a primer for the production of highly sensitive DNA probes. The system has three main steps: (i) labeling of DNA with biotinylated 11-dUTP; (ii) detection of biotinylated DNA by a one-step procedure with streptavidin-alkaline phosphatase complex; (iii) blocking of background with Tween 20. Twenty attograms (2 X 10(-17) g) of pBR322 plasmid DNA was detected by dot-blot hybridization. Upon Southern blot hybridization, 7.4 fg (7.4 X 10(-15) g) of pBR322 HindIII DNA was detected using the biotinylated pBR322 plasmid DNA probe; 40.8 ag and 7.4 fg of lambda HindIII DNA were detected with the biotinylated whole lambda DNA probe by dot and Southern blot hybridization, respectively. Specific bands were also detected with the biotinylated argininosuccinolyase probe upon Northern blotting of mouse poly(A+) RNA. Further applications for in situ hybridization are also described.     

Tissue print hybridization. A simple technique for detecting organ- and tissue-specific gene expression

We have used a new technique, which we call tissue print hybridization, to monitor organ- and tissue-specific expression of auxin-induced RNAs in soybean (Glycine max cv. Wayne) seedlings. This technique is modified from that originally published by Cassab and Varner (J Cell Biol 105: 2581–2588, 1987) for the localization of extensin protein in soybean seed using an antibody probe. We extended this original tissue print procedure by utilizing³⁵S-labeled antisense RNAs for localization of specific RNAs immobilized on nylon membranes. We also employed modifications to improve the resolution of the autoradiographic images. We have used this technique to demonstrate the tissue-specific expression of auxin-regulated genes in elongating hypocotyl regions of etiolated soybean seedlings and the rapid turn-over of RNAs encoded by these genes during gravistimulation.     

A simple and efficient method for chemical mutagenesis of DNA.

A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA segment is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragment is obtained. This methoxylamine-derivatized DNA segment is then joined with linearized M13 RF DNA, competent E. coli is transfected, and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM beta-lactamase.     

ＤＮＡ粗提取与鉴定的简易方法

介绍一种在中学可行的ＤＮＡ提取与鉴定的简易方法,该法可以不使用 离心机和一些特殊的昂贵的药品。     

A new method for detecting single nucleotide polymorphism using GFP-display

The single nucleotide polymorphism (SNP) of aldehyde dehydrogenase-2 (ALDH2) codon 487, GAA (Glu) or AAA (Lys), was examined using green fluorescent protein (GFP)-display, an electrophoretic detection method for single amino acid changes. Although no shift in migration between the GFP-ALDH (Glu487) and GFP-ALDH (Lys487) fusion proteins was observed on SDS/urea gel, the two migrated to different positions when tagged with Asp. The SNP analysis was performed with GFP-ALDH-Asp3, and GFP-ALDH-Asp3 constructed from donors having the codon GAA/GAA, GAA/AAA or AAA/AAA was detected as different patterns as expected. GFP-display is potentially a unique method in SNP analysis, which does not require any special equipment or chemicals.