Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5

High pressure liquid chromatography on the RPC-5 reversed-phase ion exchange system has been shown to have several potential applications as an initial high capacity step in the isolation of specific DNA restriction fragments. The fractionation of the Hinc II digest of lambda DNA, which contains 35 fragments with "flush ends" ranging in size from 3 x 10(6) to 7 x 10(4) daltons, has been used as a model system. Under certain conditions there are some restriction fragments whose elution relative to other fragments is different on RPC-5 chromatography than it is on gel electrophoresis. In some special circumstances it is possible to obtain satisfactory yields (60-70%) of a pure restriction fragment after a single passage through an RPC-5 column.     

A simple two-step method for efficient blunt-end ligation of DNA fragments

The formation of recombinant plasmids results from ligation between one end of the linearized vector and one end of the insert (favored by high DNA concentration), followed by self-ligation of the newly created hybrid molecule (favored by low DNA concentration). Standard protocols recommend an average DNA concentration at which both events may occur. Since this DNA concentration is not optimum for both ligation events, efficient blunt-end ligation is compromised. We describe a method for blunt-end ligation starting at a high DNA concentration for 1 h then at 1/20 the initial DNA concentration overnight. The number of recombinant plasmids obtained with this method is about 10-fold higher than with standard protocols. Restriction digestion and agarose gel electrophoresis of 10 recombinant plasmids obtained with the two-step ligation method showed that all plasmids contained one copy of the insert.     

Quick identification and localization of CpG islands in large genomic fragments by partial digestion with Hpa II and Hha I

This study examined features of patients that clinicians identified as good examples of Passive-Aggressive Personality Disorder to identify core features of the disorder and to determine which set of criteria (DSM-III-R, two definitions in the DSM-IV Options Book, or DSM-IV Negativistic) best characterized the identified patients. A national sample of licensed psychologists (N = 68) identified a patient who (based on symptoms) was a good example of Passive-Aggressive Personality Disorder. They then rated the patient on a symptom checklist composed of the Passive-Aggressive and Negativistic criteria, as well as other personality-disorder symptoms that overlap with Passive-Aggressive. Clinicians identified patients they considered exemplars for Passive-Aggressive Personality Disorder, and there was moderate consensus about their characteristic symptoms. DSM-III-R symptoms received the highest ratings, and there was little overlap with other personality disorders. Principal-component factor analysis suggested that a general pattern of passive resistance, along with a behavioral manifestation of procrastination and a second group of symptoms suggesting interpersonal difficulties, were the features of these passive-aggressive patients. More male patients were identified as good examples of the disorder, and female patients presented a more heterogeneous diagnostic picture. Implications and directions for future research are discussed, including the need to integrate research findings from the differing perspectives on personality disorders.     

DNA sequence analysis of Prinker-modified restriction fragments after collection from capillary electrophoresis with replaceable matrices

This paper demonstrates the procedure of sequencing DNA restriction fragments isolated by a recently developed fraction collector after CE separation. In particular, using pBr 322 plasmid as a model system, a double digest was performed with Eco RI and Pst 1 restriction enzymes to produce two fragments of 749 base pairs (bp) and 3612 bp, both with cohesive ends. Prinkers, specific linkers complementary to the cohesive ends, were then ligated to both fragments (increasing the size by 59 bp each). These Prinker-modified fragments were separated by CE and collected. The success of the collection was demonstrated by reinjection of each isolated fraction with laser-induced fluorescence detection, using ethidium bromide as intercalater. The 808 bp isolated fragment was then polymerase chain reaction-amplified with appropriate primers for the Prinker ends, followed by cycle sequencing. Both strands of the fragment were run on an ABI 373, sequencing 427 bases and 450 bases, respectively, with a read accuracy of 99.3%. This approach with Prinker-modified restriction fragment and automated CE fraction collection can be used as a general procedure for sequencing unknown genomic DNA as well as mutated DNA mixtures.     

A novel crucible-less inert gas atomisation method of producing titanium powder for additive manufacturing

A novel low-cost gas atomisation technology producing spherical titanium powder (wire induction heating gas atomisation, WIGA) has been developed for additive manufacturing. Combined with the gas atomisation principle, the characteristics of WIGA were analysed. The effects of the gas pressure, metal temperature and the wire-feeding speed on the particle size of the titanium powder were studied. The results indicated that the decreases in mass median particle diameter (D₅₀) and the increases in efficiency of fine size powders occurred with the increase in gas atomisation pressure and melting temperature and with the decrease of wire-feed speed. The optimum parameters are that the main gas pressure (P₀) is 4.0?MPa, the degree of superheat of the metal melt is 350°C and the wire-feed speed is 50?mm?s^?1. On the condition, the D₅₀ of titanium powder was 40.2?μm and powder morphology was spherical. Satellites rarely existed on the surface of particles.     

DNA restriction fragments length polymorphism of Mycobacterium tuberculosis isolates in Pisa, Italy

A total of 60 Mycobacterium tuberculosis strains isolated in the area of Pisa, Italy, over a period from April 1993 to December 1995, were analyzed for the IS6110-based restriction fragments length polymorphism (RFLP). Isolates were found to show a great heterogeneity and only few isolates shared identical DNA banding patterns. In particular, 55 distinct IS6110 patterns were found (average number of isolates per pattern: 1.09) and only 9 strains (15%) occurred in 4 clusters of 2-3 identical clones. Computer analysis of genetic similarities among the strains revealed a family of 17 isolates including the clustered clones implicated in recently acquired infections. No correlation was found between the RFLP DNA patterns of the isolates and drug susceptibility. Of the 5 isolates from immigrants only one showed abnormal DNA fingerprinting. Our data indicate that the patterns of M. tuberculosis isolates in Pisa area are comparable to those of countries with low-prevalence TB and that a low level of TB transmission occurs in this area.     

Competitive PCR for quantification of BM5d proviral DNA in mice with AIDS

Murine AIDS in C57BL/6 mice is caused by a unique mixture of murine leukemia viruses. We report the use of a competitive PCR to detect and quantitate BM5d proviral DNA. This assay allowed discrimination among endogenous wild-type murine retroviruses and BM5d sequences. Furthermore, the method was subsequently used to evaluate the amount of BM5d in infected mice and in infected AZT (zidovudine)-treated mice, providing an effective way to quantitatively evaluate drug efficacy in the murine AIDS model.     

Pretreatment with restriction enzyme or bovine serum albumin for effective PCR amplification of Epstein-Barr virus DNA in DNA extracted from paraffin-embedded gastric carcinoma tissue

An association between Epstein-Barr virus (EBV) and gastric carcinoma has been studied through the EBV genome present in the carcinoma cells. Recently, we found that EBV DNA in paraffin-embedded gastric carcinoma tissue was detected effectively by PCR after pretreatment of the extracted DNA with a restriction enzyme, BamHI or EcoRI. Here, we show that the PCR amplification was also enhanced by pretreatment of the DNA with other restriction enzymes or with bovine serum albumin and several other proteins. Treatment with these proteins may remove a PCR inhibitor(s) in the DNA samples extracted from the paraffin blocks.     

顺路溜井局部出矿留矿法研究

论述了适合高宝山金矿急倾斜、矿岩不稳固的薄矿脉，采用顺路溜井局部出矿留矿法试验研究成果及取得的经济效益。     

A novel method for isolation of Chlamydia pneumoniae by treatment with trypsin or EDTA

To establish a novel method for the efficient isolation of Chlamydia pneumoniae, experiments were performed to determine the effects of EDTA or trypsin treatment of C. pneumoniae on its adsorption and inclusion body formation. Treatment of C. pneumoniae with 0.1% trypsin or 1 mM EDTA significantly increased inclusion body-forming activity from 8,000- to 10,000-fold higher than that of the control. C. pneumoniae was successfully isolated in cultured cells which were inoculated with clinical specimens after treatment with 0.1% trypsin.     

DNA fingerprinting analysis by a PCR based method for monitoring the genotoxic effects of heavy metals pollution

Environmental pollutants can have deleterious effects on living organisms. At high concentrations, or at high activities, they can cause acute toxicity damaging cells, tissues and organs. Chronic toxification, on the other hand, can also cause serious damage from bio-accumulation. Plants, as biological indicators, can measure both the actual and the potential effects of pollutants, when they are used to measure effects of heavy metals. We have applied a system of "molecular fingerprinting" based on PCR (RAPD: Random Amplified Polymorphic DNA) to the evaluation of the genotoxic effects of heavy metals in order to estimate the environmental risk connected with their potential mutagenic effects in the model plant Arabidopsis thaliana, ecotype Columbia. Genomic DNA was utilised for RAPD analysis using random primers (10-mers). DNA from plants exposed to heavy metals solution displayed polymorphic bands which were not detectable in DNA of unexposed plants. The enhanced formation of RAPD polymorphisms was also observed in DNA of plant exposed in situ to an industrial pollution source. The comparison between "unexposed" and "exposed" genomes show that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.     

A novel derivatization method with 5-bromonicotinic acid N-hydroxysuccinimide for determination of the amino acid sequences of peptides

We have developed a novel method that effectively identifies the N-terminal product ions produced in the tandem mass spectrometry (MS/MS) analysis of peptides done in conjunction with the specific derivatization of the N-terminal amino group using 5-bromonicotinic acid N-hydroxysuccinimide ester (BrNA-NHS). Electrospray ionization with low-energy collision-induced dissociation (CID) MS/MS clearly differentiated the N-terminal product ions labeled with the 5-bromonicotinyl group from other ions, on the basis of the appearance of CID peaks with a doublet pattern characteristically separated by 2 mass units produced by the equal natural abundances of 79Br and 81Br. The tracing of a series of these bromine-containing product ions allows the easy amino acid sequencing of peptides. Using Gln-Arg-Leu-Gln-Ser-Asn-Gln-Leu-Lys as the test peptide, we found that within 30 minutes at pH 6.5 and 37 degrees C its alpha-amino group was completely acylated with BrNA-NHS (peptide: BrNA-NHS = 1:40; mol/mol). The epsilon-amino group of the C-terminal lysine residue was less likely to be acylated under these conditions, being only partly modified (about 20%). This suggests the possibility of keeping the epsilon-amino group free from acylation. The method was successfully applied to the determination of the amino acid sequences of peptides from porcine kidney aminoacylase I produced by digestion with lysyl endopeptidase and with Staphylococus aureus V8 protease.     

A simple method for producing a monospecific antiserum against IgM

To avoid the chemical purification of mastomys IgM, a reliable alternative method for producing a specific anti-mastomys IgM antiserum was developed. It employs the strong cross-reactivity of antisera against mouse serum proteins with mastomys serum proteins. By the immunoelectrophoresis technique, precipitates of mastomys IgM and cross-reacting rabbit anti-mouse IgM were obtained. A rabbit was immunized with these precipitates. Activity against the light chains was removed from the antiserum by passage over an immunoadsorbent column of mastomys IgG. The adsorbed antiserum was found to be specific for determinants on mastomys IgM.     

Detection of Leptospira DNA in patients with aseptic meningitis by PCR

Samples of cerebrospinal fluid from 103 patients with aseptic meningitis were tested by PCR for detection of leptospires, and the results were compared with those of the microscopic agglutination test (MAT) and an enzyme-linked immunosorbent assay for detection of immunoglobulin M (ELISA-IgM). Of these samples, 39.80% were positive by PCR and 8.74 and 3.88% were positive by MAT and ELISA-IgM, respectively.     

Inhibition of restriction endonuclease activity by DNA binding fluorochromes

Activity of type II restriction endonuclease is affected by many common factors including buffer composition and sequences flanking the recognition site (Brabec et al., Eur.J. Biochem. 216, 183, 1993). The successful development of Optical Mapping (Schwartz et al., Science, 262, 110, 1993; Meng et al., Nature Genet. 9, 432, 1995; Wang and Schwartz, PNAS, 1995 Cai et al., PNAS, 92, 5164, 1995) relied on optimization of light microscope-based imaging of fluorescently labeled DNA molecules during restriction endonuclease digestion. Little was known about the effects of commonly used DNA-fluorochromes on restriction endonuclease activity. Thus, we developed an enzyme activity assay using lambda bacteriophage DNA or adenovirus-2 DNA to evaluate the effects of five DNA binding fluorochromes (4'-6-daimidine-2-phenylindole (DAPI), ethidium bromide (EtdBr), ethidium bromide homodimer (EthD-1), bis-benzimide (H33258) and benzothiazolium-4-quinolinium dimer (TOTO-1)) on the enzymatic activities of eleven type II restriction endonucleases (Asc I, Csp I, Dra I, EcoR I, Hha I, Hind III, Not I, Rsr II, Sfi I, SgrA I and Sma I). We found that the minor groove binding fluorochrome, DAPI, did not measurably inhibit activity of this group, with the exception of Dra I. Similarly, another minor groove binding fluorochrome H33258 inhibited Dra I and Not I (slightly). The three intercalating fluorochromes EtdBr, EthD-1 and TOTO-1, however, variably inhibited the other enzymes. Since Beta-mercaptoethanol (Beta-ME) is used to discourage photodamage of stained DNA molecules, we also assessed its effect on restriction endonuclease activity. Interestingly, Dra I, Hind III, Sfi I and Sma I retained full activities at high concentration of Beta-ME (5%), but Asc I, Csp I, Not I, Rsr II and SgrA I showed varying sensitivities to the Beta-ME. Isoschizomers Csp I and Rsr II behaved differently to both fluorochromes and Beta-ME. The results presented here should provide a basis for further development of new Optical Mapping-based techniques requiring fluorescence labeling of other actively imaged enzymatic reactions.