Detection of a few picograms of DNA on polyacrylamide gels by silver staining

DNA fragments separated on polyacrylamide gels are silver stained in ethanolamine solution. The staining procedure can be completed in 3 1/2 h. Illumination of the gels on a black background increases the sensitivity of detection compared with the usual transillumination. The limit level of detection is 3-5 pg per band with a cross-sectional area of 5 mm2. Five to fifty picograms of DNA may be detected quantitatively by scanning the gels. The method will detect 0.1 to 1 ng per band of low-molecular-weight RNA components.     

Detection of Pseudomonas aeruginosa from clinical and environmental samples by amplification of the exotoxin A gene using PCR.

PCR was used to detect Pseudomonas aeruginosa from water samples by amplifying a 396-bp region of the exotoxin A (ETA) structural gene sequence. The identify of the amplified 396-bp fragment was confirmed by digesting it with PvuI restriction endonuclease, which produced the predicted 246- and 150-bp fragments. Specific primers amplified ETA-positive P. aeruginosa DNA, whereas other species of Pseudomonas and GC-rich bacteria did not yield any 396-bp fragment. The specificity and sensitivity of the assay were 100 and 96%, respectively, which confirms the assay's reliability for diagnostic and epidemiological studies. The assay can detect as few as 5 to 10 cells in a 10-ml water sample or 0.1 pg of P. aeruginosa DNA per reaction mixture (5 microliters) by ethidium bromide staining of an agarose gel. Ten-times-lower concentrations were detected by hybridization with a digoxigenin-labeled oligonucleotide probe internal to the PCR product. With this PCR method, ETA-positive P. aeruginosa was detected in animal cage water samples at a level of 40 cells per ml. This method is rapid and less cumbersome than other diagnostic methods for the identification of P. aeruginosa strains. The method described can be used to detect a low level of P. aeruginosa from environmental and clinical samples without the use of selective media or additional biochemical tests.     

Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

The fast diagnosis of Mycoplasma primary atypical pneumonia is impaired by the lack of routinely available culture methods for isolation of Mycoplasma pneumoniae from clinical specimens. Likewise, serological methods commonly used for diagnosis are insensitive and non-specific. In this study, we have established and applied the polymerase chain reaction (PCR) technique to detect M. pneumoniae DNA in clinical samples originating from the respiratory tract. The PCR results were compared with those from culture and serology tests. To standardize the detection of M. pneumoniae by PCR, we first used DNA from culture grown organisms and clinical samples seeded with M. pneumoniae. PCR amplification was performed with M. pneumoniae-specific primers to amplify 144, 153 and 631 bp DNA fragments by using primer pairs MP5-1/MP5-2, P1-178/P1-331 and P1-178/P1-809, respectively. The amplification of the 631 bp DNA fragment was found to be most sensitive for the detection of M. pneumoniae. Using the most sensitive PCR, a total of 47 respiratory specimens from patients suspected of community acquired pneumonia were tested. While none of the specimens were positive for M. pneumoniae in culture, 6 specimens gave positive results by PCR. In 4 out of the 5 PCR positive samples tested serologically, the results were supported by elevated levels of anti-mycoplasma IgG/IgM/IgA. Thus, these results suggest that PCR is the most sensitive method to detect M. pneumoniae in clinical specimens.     

A RAPD marker associated with B chromosomes in Partamona helleri (Hymenoptera, Apidae)

The hymenopteran Partamona helleri is found in southwestern Brazil in the Mata Atlantica from the north of the state of Santa Catarina until the south of Bahia. This work shows that P. helleri can carry up to four B chromosomes per individual. In order to obtain more information about P. helleri B chromosomes, the RAPD technique was used to detect DNA fragments associated with these chromosomes. The results showed that the RAPD technique is useful to detect specific sequences associated with B chromosomes. One RAPD marker was identified, cloned and used as probe in a DNA blot analysis. This RAPD marker hybridized with sequences present only in individuals containing B chromosomes.     

A new procedure for determining thymine residues in DNA sequencing. Photoinduced cleavage of DNA fragments in the presence of spermine

A new procedure for T specific cleavage of DNA fragments utilizing photoreaction with spermine has been described. Irradiation of 3'-[32P]-end-labeled DNA fragments for 10-20 min with a germicidal lamp emitting mainly 254-nm light in the presence of 1 M spermine in distilled water resulted in a T specific cleavage of the DNA chains. This method does not require piperidine treatment. By contrast, when the DNA fragments were irradiated in the presence of methylamine under similar conditions, both G and T bands with the intensity of G greater than T have appeared. A similar but less selective T cleavage has also been observed in the irradiation of 5'-[32P]-end-labeled DNA fragments in the presence of spermine followed by brief heating of the photolysate in a loading buffer for gel electrophoresis. The T specific photoreaction with spermine and the G greater than T reaction with methylamine described here may be conveniently used in combination with the standard Maxam-Gilbert's reactions to provide independent confirmatory readings.     

Yields of damage to C4' deoxyribose and to pyrimidines in pUC18 by the direct effect of ionizing radiation

Our mechanistic understanding of damage formation in DNA by the direct effect relies heavily on what is known of free radical intermediates studied by EPR spectroscopy. Bridging this information to stable product formation requires methods with comparable sensitivities, a criterion met by the (32)P-post-labeling assay developed by Weinfeld and Soderlind, [Weinfeld,M. and Soderlind,K.-J.M. (1991) (32)P-Postlabeling detection of radiation-induced DNA damage: identification and estimation of thymine glycols and phosphoglycolate termini. Biochemistry, 30, 1091-1097] which when applied to the indirect effect, detected phosphoglycolate (pg) and thymine glycol (Tg). Here we applied this assay to the direct effect, measuring product yields in pUC18 films with hydration levels (Γ) of 2.5, 16 or 23 waters per nucleotide and X-irradiated at either 4 K or room temperature (RT). The yields of pg [G(pg)] for Γ ≈ 2.5 were 2.8 ± 0.2 nmol/J (RT) and 0.2 ± 0.3 nmol/J (4 K), which is evidence that the C4' radical contributes little to the total deoxyribose damage via the direct effect. The yield of detectable base damage [G(B*)] at Γ ≈ 2.5 was found to be 30.2 ± 1.0 nmol/J (RT) and 12.9 ± 0.7 nmol/J (4 K). While the base damage called B*, could be due to either oxidation or reduction, we argue that two reduction products, 5,6-dihydrouracil and 5,6-dihydrothymine, are the most likely candidates.     

Detection of the catalytic activities of DNA polymerases and their associated exonucleases following SDS-polyacrylamide gel electrophoresis.

A method is described to detect DNA polymerases and nucleases in homogeneous or crude enzyme preparations after electrophoresis in SDS-polyacrylamide gels(2) containing the appropriate template or substrate. DNA polymerases are electrophoresed in a gel containing gapped calf thymus DNA and after a renaturation treatment, the gel is incubated in a reaction mixture in which one deoxyribonucleoside triphosphate is [alpha-32P]-labelled. Incorporation of radioactivity into DNA is detected at the vicinity of the polymerase band by autoradiography. An associated nuclease activity can be measured after electrophoresis in a gel containing 32P-labelled gapped DNA, when nucleolytic digestion is seen as a clear band in the resulting autoradiogram. The gels can subsequently be stained with Coomassie blue to establish identical molecular weights of polymerase, nuclease and protein bands. Applications of this technique are discussed.     

Sensitive detection of Mycoplasma pulmonis by using the polymerase chain reaction

Detection of Mycoplasma pulmonis was examined by using the polymerase chain reaction (PCR) for amplifying a specific DNA sequence. In gel electrophoresis which was conducted to detect the amplified products, only 1 pg of M. pulmonis DNA could be detected following 30 cycles of amplification, while no amplified product was detected even from 1 microgram of M. arthritidis or M. neurolyticum DNA. Furthermore, 10 colony-forming units of M. pulmonis could be detected by direct amplification from the mycoplasma suspension. These results suggest the usefulness of the PCR as a highly sensitive, specific, and rapid method for direct detection of M. pulmonis.     

Characteristics of structures of Drosophila polytene chromosomes formed by transposable DNA fragments

An electron microscopic analysis of regions of Drosophila melanogaster polytene chromosomes into which DNA fragments of different genetic composition were inserted by the P element-mediated transformation was performed. In 4 of 5 regions studied with integrated DNA sequences of the hsp28-ry, hsp70-Adh, ry-hsp70-beta-gal genes new bands appeared. Apparently their generation is mainly caused by integration of the DNA fragments in interbands. Absence of a new band in transformed region in one of the stocks can be explained by fusion of the insertion with a band existed in the initial untransformed stock. Among the transformants studied, the minimum length of DNA fragment revealed as a new band is about 5 kb. DNA packing ratio of such the bands varies from 30 to 50. The activation of the inserted genes by heat shock allows to trace peculiarities of the new bands puffing. The puff sizes correlate with the length of the activated genes. If the DNA of the fragment consists of the sequence of one gene, its activation will lead to decondensation of the whole band. In the case when DNA fragment consists of 2 genes and the promoter of activated gene is situated inside the sequence, the band is splitted after gene activation at the beginning and then separated portion of the band is decondensed and puffed. The data obtained evidence that a band of polytene chromosome is not a unit of decondensation. DNA packing ratio in puffs is equal to 1.5-3.5.     

Rapid identification of Arabidopsis insertion mutants by non-radioactive detection of T-DNA tagged genes

To assist in the analysis of plant gene functions we have generated a new Arabidopsis insertion mutant collection of 90 000 lines that carry the T-DNA of Agrobacterium gene fusion vector pPCV6NFHyg. Segregation analysis indicates that the average frequency of insertion sites is 1.29 per line, predicting about 116 100 independent tagged loci in the collection. The average T-DNA copy number estimated by Southern DNA hybridization is 2.4, as over 50% of the insertion loci contain tandem T-DNA copies. The collection is pooled in two arrays providing 40 PCR templates, each containing DNA from either 4000 or 5000 individual plants. A rapid and sensitive PCR technique using high-quality template DNA accelerates the identification of T-DNA tagged genes without DNA hybridization. The PCR screening is performed by agarose gel electrophoresis followed by isolation and direct sequencing of DNA fragments of amplified T-DNA insert junctions. To estimate the mutation recovery rate, 39 700 lines have been screened for T-DNA tags in 154 genes yielding 87 confirmed mutations in 73 target genes. Screening the whole collection with both T-DNA border primers requires 170 PCR reactions that are expected to detect a mutation in a gene with at least twofold redundancy and an estimated probability of 77%. Using this technique, an M2 family segregating a characterized gene mutation can be identified within 4 weeks.     

Hybridization of labeled RNA to DNA in agarose gels.

Specific DNA restriction endonuclease fragments can be identified after electrophoresis in agarose gels by hybridization in the gel (in situ) to radioactive homologous RNA. RNA-DNA hybrids are detected by autoradiography of the gel. Comparison of band patterns of the autoradiogram and the ethidium bromide stained gel allows the identification of the DNA fragment which is complementary to the RNA probe. The technique is rapid, easy and inexpensive. It is sensitive enough to detect individual genes in a mixture of fragments produced by restriction enzyme digestion of complex cellular DNA. We have used this technique to determine which of the Hin III and Eco R1 fragments of phi80d3ilv+su+7 and E. coli DNAs contain the 5S, 16S and 23S ribosomal RNA (rRNA) genes of E. coli.     

Fractionation of rat ventricular nuclei.

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.     

Agarose gel electrophoretic evidence for domains of nuclear DNA linked with bonds cleavable with sulfhydryl molecules

Complexes of intact nuclear DNA with proteins undissociable by 2.0 M NaCl and nonionic detergents were analyzed by agarose gel electrophoresis following physical or enzymatic fragmentation. Sulfhydryl molecules converted these DNAs (but not the bacteriophage lambda DNA) into smaller-Mr forms. Following limited restriction endonuclease digestion of complexes with PstI most of the nuclear DNA formed a high-molecular-mass band in the 60-110 kbp range. These 60-110 kbp fragments, releasable from the rest of nuclei by sulfhydryl molecules, have similar sizes to nuclear DNA loops detected by other techniques and may derive from supranucleosomal organizational units in the chromatin complex.     

Detection and mapping of homologous, repeated and amplified DNA sequences by DNA renaturation in agarose gels.

A new molecular hybridization approach to the analysis of complex genomes has been developed. Tracer and driver DNAs were digested with the same restriction enzyme(s), and tracer DNA was labeled with 32P using T4 DNA polymerase. Tracer DNA was mixed with an excess amount of driver, and the mixture was electrophoresed in an agarose gel. Following electrophoresis, DNA was alkali-denatured in situ and allowed to reanneal in the gel, so that tracer DNA fragments could hybridize to the driver only when homologous driver DNA sequences were present at the same place in the gel, i.e. within a restriction fragment of the same size. After reannealing, unhybridized single-stranded DNA was digested in situ with S1 nuclease. The hybridized tracer DNA was detected by autoradiography. The general applicability of this technique was demonstrated in the following experiments. The common EcoRI restriction fragments were identified in the genomes of E. coli and four other species of bacteria. Two of these fragments are conserved in all Enterobacteriaceae. In other experiments, repeated EcoRI fragments of eukaryotic DNA were visualized as bands of various intensity after reassociation of a total genomic restriction digest in the gel. The situation of gene amplification was modeled by the addition of varying amounts of lambda phage DNA to eukaryotic DNA prior to restriction enzyme digestion. Restriction fragments of lambda DNA were detectable at a ratio of 15 copies per chicken genome and 30 copies per human genome. This approach was used to detect amplified DNA fragments in methotrexate (MTX)-resistant mouse cells and to identify commonly amplified fragments in two independently derived MTX-resistant lines.     

Specific detection of Plasmodium falciparum malaria by a molecularly cloned DNA probe

A highly repeated DNA sequence from Plasmodium falciparum was cloned and used as a probe in molecular hybridization to detect malaria. Our results indicate that the probe is specific to P. falciparum but not to other species of Plasmodium and is extremely sensitive. As little as a 20 pg parasite DNA, which is equivalent to about 1000 parasites can be detected. The cloned DNA can be used as a diagnostic tool to follow the course of infection of falciparum malaria.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

A non-radioactive automated method for DNA sequence determination

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.     

Using lambda exonuclease inhibition assays to map carcinogen binding sites.

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.     

Detection of Chlamydia trachomatis in clinical specimens by nucleic acid spot hybridization

A nucleic acid spot hybridization assay was used to detect Chlamydia trachomatis DNA. The hybridization probes included DNA isolated from elementary bodies of lymphogranuloma venereum (LGV) strains and cloned fragments of both chromosomal and plasmid DNA. The sensitivity of the test was in the range 10 to 100 pg homologous DNA and 10 in vitro infected cells. Cross-reactivity with bacterial DNA was avoided when purified chlamydia-specific DNA fragments were used as probes. C. trachomatis was detectable in most of the clinical specimens with large amounts of infectious particles. Also some isolation-negative specimens gave a positive signal in the test.     

Comparison of PCR-RFLP with allele-specific PCR in genetic testing for spinal muscular atrophy

PCR-based methods for the detection of homozygous deletion of exon 7 of the SMN1 gene have been widely used in genetic testing for spinal muscular atrophy (SMA). We compared the most commonly used PCRrestriction fragment length polymorphism (PCR-RFLP) assay with an allele-specific PCR method, evaluating their potential application in direct testing, prenatal prediction, and preimplantation diagnosis, in terms of a range of DNA amounts used in such testing. We showed that PCR-RFLP could identify the SMN1 exon 7 by amplifying 10 pg of genomic DNA, and could differentiate SMN1 from SMN2 at the 100-pg DNA level (DraIdigested SMN2 fragments served as an internal control for PCR efficiency). In contrast, allele-specific PCR for SMN1, despite some advantages in a rapid preimplantation diagnosis, quickly lost its specificity when 100 pg of genomic DNA was used. In addition, the absence of a SMN1 fragment at the 10-pg DNA level may be due to a PCR amplification failure, and, thus, it is difficult to interpret without a proper internal control. Our data indicate that PCR-RFLP can be used for most diagnostic purposes, whereas the use of allelespecific PCR may be considered with caution under certain circumstances.