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Related Experiment Videos

Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

P Humphries, R Old, L W Coggins

    Nucleic Acids Research
    |March 1, 1978
    PubMed
    Summary
    This summary is machine-generated.

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    Researchers synthesized double-stranded DNA complementary to Xenopus globin messenger RNA (mRNA) using enzymes. This DNA was cloned into plasmids in E. coli, creating DNA copies of the globin gene.

    Area of Science:

    • Molecular Biology
    • Biochemistry
    • Genetics

    Background:

    • Messenger RNA (mRNA) serves as a template for protein synthesis.
    • Complementary DNA (cDNA) synthesis is crucial for gene cloning and manipulation.
    • Xenopus laevis is a model organism in developmental biology.

    Purpose of the Study:

    • To synthesize double-stranded DNA complementary to Xenopus globin mRNA in vitro.
    • To clone and amplify these DNA sequences using bacterial plasmids.
    • To confirm the successful integration and presence of globin gene sequences.

    Main Methods:

    • In vitro synthesis using reverse transcriptase, E. coli DNA polymerase I, and S1 endonuclease.
    • Size selection of synthesized DNA via neutral sucrose gradient sedimentation.

    Related Experiment Videos

  • Oligonucleotide tailing with terminal transferase and plasmid insertion.
  • Bacterial transformation (transfection) and plasmid amplification.
  • Hybridization techniques (in situ and solution) for sequence identification.
  • Gel electrophoresis and heteroduplex mapping for DNA analysis.

    • Main Results:

    • Successfully synthesized double-stranded DNA molecules complementary to Xenopus globin mRNA.
    • Extended DNA termini and integrated them into pCR1 plasmid DNA.
    • Amplified recombinant plasmids in E. coli.
    • Confirmed the presence of Xenopus globin sequences in recombinant plasmids through various hybridization and mapping methods.

    Conclusions:

    • Demonstrated a method for in vitro synthesis and cloning of Xenopus globin cDNA.
    • Established recombinant plasmids containing extensive DNA copies of Xenopus globin mRNA.
    • Provided a foundation for further studies on globin gene expression and regulation.