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Related Experiment Videos

Oligonucleotide capture during end joining in mammalian cells.

D B Roth1, G N Proctor, L K Stewart

  • 1Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030.

Nucleic Acids Research
|December 1, 1991
PubMed
Summary

Filler DNA in genetic rearrangements may originate from oligonucleotide fragments. Researchers tested this by microinjecting oligonucleotides with SV40 genomes, finding only complementary double-stranded DNA was incorporated.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Cell Biology

Background:

  • Illegitimate recombination in mammalian cells can result in genetic rearrangements with extra nucleotides, known as filler DNA.
  • A hypothesis suggests filler DNA arises from oligonucleotide fragments joining broken DNA ends before ligation.

Purpose of the Study:

  • To investigate whether oligonucleotide fragments can be incorporated as filler DNA during genetic rearrangements.
  • To determine the role of complementarity in the incorporation of oligonucleotides into broken DNA ends.

Main Methods:

  • Microinjection of SV40 genomes linearized in the T antigen intron with defined single-stranded and double-stranded oligonucleotides.
  • Screening of viable genomes for incorporation of unique restriction sites from injected oligonucleotides.

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  • Nucleotide sequencing of junctions from selected genomes to analyze filler DNA origins.

    • Main Results:

    • Double-stranded oligonucleotides fully complementary to the SV40 DNA ends were readily incorporated.
    • Incorporation of non-complementary or single-stranded oligonucleotides was not detected by restriction analysis.
    • Sequence analysis of two genomes with filler DNA from non-complementary oligonucleotide co-injection did not unambiguously link the filler DNA to the injected oligonucleotides.

    Conclusions:

    • Complementary double-stranded oligonucleotides can be incorporated into linear DNA molecules during illegitimate recombination.
    • The origin of filler DNA in illegitimate recombination remains largely unclear, as non-complementary oligonucleotides were not demonstrably incorporated.
    • Further research is needed to elucidate the precise mechanisms and sources of filler DNA during genetic rearrangements.