Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction
| Autor: | R C Greene, R B Blitchington, Kenneth H. Wilson |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
DNA
Bacterial Microbiology (medical) Genetics Base Sequence Inverse polymerase chain reaction Hybridization probe Molecular Sequence Data Gene Amplification Biology Ribosomal RNA 16S ribosomal RNA DNA Ribosomal Polymerase Chain Reaction Microbiology law.invention chemistry.chemical_compound chemistry law RNA Ribosomal 16S DNA Probes Ribosomal DNA DNA Polymerase chain reaction Research Article Southern blot |
| Zdroj: | Journal of Clinical Microbiology. 28:1942-1946 |
| ISSN: | 1098-660X 0095-1137 |
| DOI: | 10.1128/jcm.28.9.1942-1946.1990 |
| Popis: | The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens. |
| Databáze: | OpenAIRE |
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