Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have developed a novel plasmid isolation procedure and have adapted it for use on an automated nucleic acid extraction instrument. The protocol is based on the finding that phenol extraction of a 1 M guanidinium thiocyanate solution at pH 4.5 efficiently removes genomic DNA from the aqueous phase, while supercoiled plasmid DNA is retained in the aqueous phase. S1 nuclease digestion of the removed genomic DNA shows that it has been denatured, which presumably confers solubility in the organic phase. The complete automated protocol for plasmid isolation involves pretreatment of bacterial cells successively with lysozyme, RNase A, and proteinase K. Following these digestions, the solution is extracted twice with a phenol/chloroform/water mixture and once with chloroform. Purified plasmid is then collected by isopropanol precipitation. The purified plasmid is essentially free of genomic DNA, RNA, and protein and is a suitable substrate for DNA sequencing and other applications requiring highly pure supercoiled plasmid.
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PMID:Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol. 171 49

Routine methods of extraction of DNA from blood involve the enrichment of cells by Ficoll-Hypaque gradient centrifugation followed by lysis of the cells with extraction buffer, proteinase K digestion of the lysate, and phenol:chloroform-isoamyl alcohol extraction. These methods generally require large amounts of blood, which poses a problem with pediatric patients. To overcome this, we developed a new method of extracting DNA directly from whole blood. This method involves the treatment of whole blood with an equal volume of NaI (3 M final concentration) followed by chloroform:isoamyl alcohol extraction to clear hemoglobin and cell debris. The clear aqueous layer is then mixed with isopropanol to obtain DNA. A large number of samples can easily be handled by this extraction procedure, as it can be carried out in 30 min and requires only a microcentrifuge.
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PMID:An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents. 195 87

Two methods were compared for the extraction of DNA from small numbers of bacterial cells. The first method involved lysis of cells with SDS in the presence of proteinase K, treatment with hexadecyltrimethyl ammonium bromide (CTAB) and precipitation of DNA with isopropanol. In the second method, DNA was extracted by treatment of the cells with guanidine hydrochloride (GHCl) and precipitated with ethanol. Thirty strains of representative gram positive and gram negative species were included in the study. Preparations derived from confluent growth on one-quarter of the surface of agar plates and from 10(8) cells were subjected to each extraction procedure and analyzed for their content of DNA, RNA and protein. The suitabilities of the resultant DNA for restriction enzyme digestion and biotin-labelling by a random primer technique were also assessed. In general, the CTAB method yielded greater amounts of DNA than the GHCl procedure. RNA was present in most preparations of both types, but in amounts detectable only by agarose gel electrophoresis. The latter technique also revealed that DNA was not excessively sheared by either procedure. Protein was detected in some CTAB and GHCl preparations, but was not consistently associated with one or the other method. DNA obtained by both methods could be digested by the restriction enzyme EcoR I. In addition, biotin-labelled DNA probes prepared from CTAB and GHCl preparations were capable of hybridizing with homologous target DNA fixed to nitrocellulose. Since the CTAB method was consistently successful in recovering DNA from preparations containing 10(8) cells, it may be more suitable for the direct treatment of single colonies taken from primary isolation plates or plaque samples.
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PMID:Comparison of two methods for the small-scale extraction of DNA from subgingival microorganisms. 263 97

A new, rapid procedure for purifying bacterial plasmids with high recovery is described. The sequence of operations consists essentially of treatment with alkali, ribonuclease, and proteinase K, followed by chisam extraction and gel filtration on Sephacryl S-1000, and finally a precipitation step using isopropanol at room temperature. The method gives rather good yields of plasmid DNA of high purity, and lends itself to scaling up.
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PMID:A novel chromatographic procedure for purification of bacterial plasmids. 631 36

Native DNA molecules isolated either in the presence of 50 micrograms x ml(-1) of proteinase K (PK-DNA) or in the presence of 6 mg x ml(-1) autodigested pronase (PRO-DNA) are about equal in size. Since shear forces were avoided as far as possible during the isolation procedure, the largest molecules found were longer than 100 microns. The average length of the traced molecules was 34.2 microns for PK-DNA and 29.7 microns for PRO-DNA. In contrast to PK-DNA the length of PRO-DNA molecules undergoes a dramatic change during denaturation. The average contour length of a denatured PRO-DNA molecules is only 6.9 microns. This reduction in length cannot be explained by shrinkage due to changes in ionic strength, pH and the effect of denaturing agents. Moreover, PK-DNA identically denatured was not dramatically changed in size. From this it must be concluded that PRO-DNA contains more internal ends than PK-DNA. This conclusion is supported by the results indicating that PRO-DNA is much more sensitive to nuclease S1 than PK-DNA. The results are consistent with previously published biochemical data suggesting that chromosomal DNA is 'nicked' or 'gapped' in a protease-catalyzed reaction at distinct protease-sensitive sites.
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PMID:Size of native and denatured DNA of Ehrlich ascites tumour cells isolated in the presence of different protease concentrations. 701 43

Two methods for the successful extraction of DNA from foods are described. The rapid lysis method uses a proteinase K buffer system to lyse cells and solubilize food samples. DNA is then precipitated using isopropanol. The second method achieves cell lysis using toluene and mutanolysin, and solubilization using guanidium thiocyanate. Following protein removal with organic solvents DNA is precipitated with isopropanol. Both methods enabled the polymerase chain reaction to be applied directly to DNA extracted from samples of cheese, coleslaw and raw chicken and allowed the direct rapid, sensitive and specific detection of Yersinia enterocolitica, Aerococcus viridans and Listeria monocytogenes in these foods.
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PMID:The direct application of the polymerase chain reaction to DNA extracted from foods. 776 15

The lack of simple and efficient methods for extraction of DNA from Nocardia spp. has hampered molecular manipulation of the DNA for diagnostic purposes. In the present study, a method for the rapid extraction of undegraded genomic nocardial DNA was established. Briefly, 14 pathogenic Nocardia strains were grown at 37 degrees C for 3 to 5 days in Sauton broth containing 0.05% Tween 80. Subsequently, the cultures were treated for 48 h with 1.2 mg of cycloserine per ml (final concentration). Cells were then harvested by centrifugation and treated with a lysis solution containing 3 mg of lysozyme per ml. This was followed by the addition of proteinase K and sodium dodecyl sulfate to final concentrations of 0.2 mg/ml and 0.5%, respectively, and incubation for 1 h at 50 degrees C. DNA was precipitated with isopropanol after phenol-chloroform-isoamyl alcohol extractions and RNase treated before being quantitated and analyzed by agarose gel electrophoresis. The average undegraded DNA yields obtained were 101 micrograms for Nocardia brasiliensis and 121 micrograms for N. asteroides. This DNA was suitable for restriction endonuclease digestion and PCR amplification, which are methods being applied to the characterization and diagnosis of slowly growing organisms such as Nocardia spp.
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PMID:A rapid and gentle method for isolation of genomic DNA from pathogenic Nocardia spp. 887 44

Lactobacillus amylovorus LMG P-13139, isolated from corn steep liquor, produces two bactericidal peptides with respective estimated molecular masses of 4.5 and 6.0 kDa upon denaturing sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The antimicrobial activity detected in the fermentation supernatant fraction of L. amylovorus LMG P-13139 was heat stable (20 min, 121 degrees C), displayed a narrow inhibitory spectrum, and was sensitive to proteinase K, trypsin, and alpha-chymotrypsin but insensitive to alpha-amylase, lysozyme, catalase, and lipase. The 4.5-kDa bacteriocin was purified and characterized and designated lactobin A. Lactobin A was isolated as a floating pellicle from culture supernatant brought to 35% saturation with ammonium sulfate. Upon this ammonium sulfate treatment, crude lactobin A was incorporated, together with Tween 80 as a major contaminant, in high-molecular-mass complexes sized at approximately 670 kDa by gel filtration chromatography. Contaminating fatty acids were removed from these micelles by a simple one-step methanol-chloroform extraction without loss of activity. Both inhibitory peptides were separated in an isocratic isopropanol gradient on a PepRPC 5/5 reversed-phase column, and both peptides retained activity towards Lactobacillus helveticus ATCC 15009 upon separation. Lactobin A has a molecular mass determined by electrospray mass spectrometry of 4,879 +/- 0.69 Da. Its peptide chain contains 50 unmodified amino acids, of which 26% are glycine residues and 40% are hydrophobic residues (A, V, L, I, and P). It displays the highest structural homology (42% identity and 28% similarity) with the lafX gene product, encoded by the second open reading frame of the lactacin F operon. These data strongly indicate that lactobin A belongs to the class IIb bacteriocins according to the classification of Klaenhammer.
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PMID:Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139. 897 34

Background: RNA is extensively degraded by routine formalin fixation to fragments averaging 200 nucleotides (nt). Several methods for the recovery of amplifiable RNA from formalin-fixed, paraffin-embedded tissue have been described; however, a universally accepted approach in a clinical molecular diagnostic laboratory has not yet emerged. Methods and Results: Amplifiable RNA can be recovered with high efficiency from all types of formalin-fixed, paraffin-embedded tissue using proteinase K digestion, either a phenol-chloroform or an acidic guanidinium thiocyanate-phenol chloroform extraction step, and isopropanol precipitation in the presence of glycogen. Designing primers to detect a small target was critical for consistent RNA amplification in the following assays, with the target size indicated: hepatitis C virus, 169nt; morbillivirus, 78 nt; influenza virus, 113 nt; the npm-alk fusion product resulting from t(2;5) translocation, 175 nt; and the bcr-abl fusion product resulting from t(9;22) translocation, 93 or 168 nt. Conclusions: With use of beta-2-microglobulin as the control messenger RNA target for assessing the recovery of amplifiable RNA from human tissue, amplifiable RNA was recovered from 216 of 225 blocks (96%). In a series of veterinary specimens, which were largely postmortem and moderately to severely autolyzed, 158 of 199 blocks (79%) yielded amplifiable RNA using a beta-actin target. Amplifiable influenza RNA has been recovered from archival paraffin blocks as old as 79 years.
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PMID:Optimization of the Isolation and Amplification of RNA From Formalin-fixed, Paraffin-embedded Tissue: The Armed Forces Institute of Pathology Experience and Literature Review. 1046 13

The aim of this study was to develop a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the analysis of benzodiazepines in human hair. The method was tested by analyzing hair samples from forensic and clinical psychiatric patients where benzodiazepines had been prescribed during hospitalization and after care. Hair samples were obtained at discharge from the clinic and then after six months. Two-centimeter segments of the hair samples (10-30 mg) were washed once with isopropanol, three times with phosphate buffer, and again with isopropanol, dried, weighed, and digested with proteinase K before solid-phase extraction with BondElut Certify columns. Diazepam, nordiazepam, oxazepam, alprazolam, OH-alprazolam, nitrazepam, 7-aminonitrazepam, flunitrazepam, 7-aminoflunitrazepam, clonazepam, and 7-aminoclonazepam were quantitated in MRM mode using one transition for each analyte and deuterated internal standard. The calibration range was 0.125-5 ng/mg for diazepam, nordiazepam, and oxazepam and 0.025-1.0 ng/mg for the other compounds. In the hair samples analyzed, diazepam, flunitrazepam, nitrazepam, and clonazepam was detected together with their metabolites. Alprazolam was not detected in any sample. Segmental hair analysis revealed differences in drug deposition in hair before and after release from psychiatric treatment. Both increases and decreases of hair drug concentrations were seen after release even though the prescribed dose was the same. This was taken as an indication of noncompliance during the after-care period. We conclude that the extraction and LC-MS-MS procedures were adequate to detect benzodiazepines in hair and that the results indicated that segmental hair analysis might provide retrospective information about medication intake.
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PMID:Segmental ion spray LC-MS-MS analysis of benzodiazepines in hair of psychiatric patients. 1242 3


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