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

Presently genetic analyses for thalassemia types require relatively large amounts of heparinized blood (5 to 10 ml), and transport as well as degeneration of these sample is a problem in the developing world. We have developed a new method to simplify this procedure and obtain DNAs from small specimens. As experimental materials, thinly smeared blood on a glass slide or blood filtered with and adhered on polysthylene telephtalate (PST) fibers were used. These materials could be safely stored without interfering with DNA extraction for up to 3 months. The slide materials were digested with proteinase K, and DNA was extracted with Tris-EDTA-phenol:chloroform and precipitated with absolute ethanol. The PST specimens were washed with physiologic saline and treated in the same manner as described above. Products were easily amplified by PCR and digested with restriction endonucleases for beta thalassemia typing as well as for HLA-DQA1 gene typing. Results obtained by this method correlated well with previously reported incidences for thalassemia and HLA-DQA1 types in Thailand. This method can be used in the routine laboratory because it allows for stable and biosafe genetic analyses.
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PMID:Detection of thalassemia genes using smeared blood film or leukocytes adhering to polysthylene fibers. 964 Jun 14

The typing of nuclear DNA from hair shafts has often been unsuccessful to date. We tried to type one of the nuclear DNA loci, HLA-DQA1, from hair shafts, using an efficient cetyl-trimethyl ammonium bromide (CTAB) precipitation for DNA purification and a sensitive semi-nested PCR. After thorough washing with ethanol and water, hair shafts were digested by proteinase K in the presence of dithiothreitol, followed by a purification step including CTAB-DNA precipitation. The specific region of HLA-DQA1 gene was amplified by the semi-nested PCR, and the amplified products were cloned and sequenced. The HLA-DQA1 genotype was determined by comparing the sequence to the known sequence of each allele. All genotypes of HLA-DQA1 were successfully typed with hair shafts from six known heterozygotes, although one of them showed the predominant appearance of one allele. For correct typing, a template DNA equivalent to a hair shaft of 5 or 10 cm in length was necessary. Without the CTAB-DNA precipitation step, DNA extract from such hair shafts inevitably contains enough melanin to inhibit PCR. The present results suggest that hair shafts can be used for the typing of nuclear DNA loci.
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PMID:Purification of nuclear DNA from single hair shafts for DNA analysis in forensic sciences. 1293 96