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Locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification.

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  • المؤلفون: Sun Z;Sun Z; Chen Z; Hou X; Li S; Zhu H; Qian J; Lu D; Liu W
  • المصدر:
    PloS one [PLoS One] 2008; Vol. 3 (11), pp. e3701. Date of Electronic Publication: 2008 Nov 11.
  • نوع النشر :
    Journal Article; Research Support, Non-U.S. Gov't
  • اللغة:
    English
  • معلومة اضافية
    • المصدر:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • بيانات النشر:
      Original Publication: San Francisco, CA : Public Library of Science
    • الموضوع:
      Genome, Bacterial*; DNA Primers/*chemistry ; Oligonucleotides/*chemistry ; Polymerase Chain Reaction/*methods; DNA, Bacterial/chemistry ; Genotype ; Kinetics
    • نبذة مختصرة :
      Background: Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can save a lot of time, cost and labor compared to traditional single reaction detection methods. However, the multiplexing method currently used requires precise handiwork and many complicated steps, making a new, simpler technique desirable. Oligonucleotides containing locked nucleic acid residues are an attractive tool because they have strong affinities for their complementary targets, they have been used to avoid dimer formation and mismatch hybridization and to enhance efficient priming. In this study, we aimed to investigate the use of locked nucleic acid pentamers for genomic DNA amplification and multiplex genotyping.
      Results: We designed locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification. The locked nucleic acid pentamers were able to prime amplification of the selected sequences within the investigated genomes, and the resulting products were similar in length to those obtained by restriction digest. In Real Time PCR of genomic DNA from three bacterial species, locked nucleic acid pentamers showed high priming efficiencies. Data from bias tests demonstrated that locked nucleic acid pentamers have equal affinities for each of the six genes tested from the Klebsiella pneumoniae genome. Combined with suspension array genotyping, locked nucleic acid pentamer-based PCR amplification was able to identify a total of 15 strains, including 3 species of bacteria, by gene- and species-specific probes. Among the 32 species used in the assay, 28 species and 50 different genes were clearly identified using this method.
      Conclusion: As a novel genomic DNA amplification, the use of locked nucleic acid pentamers as universal primer pairs in conjunction with suspension array genotyping, allows for the identification of multiple distinct genes or species with a single amplification procedure. This demonstrates that locked nucleic acid pentamer-based PCR can be utilized extensively in pathogen identification.
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    • الرقم المعرف:
      0 (DNA Primers)
      0 (DNA, Bacterial)
      0 (Oligonucleotides)
      0 (locked nucleic acid)
    • الموضوع:
      Date Created: 20081113 Date Completed: 20081212 Latest Revision: 20211020
    • الموضوع:
      20221213
    • الرقم المعرف:
      PMC2577006
    • الرقم المعرف:
      10.1371/journal.pone.0003701
    • الرقم المعرف:
      19002243