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An improved DNA sequencing strategy.

H C Lin, S P Lei, G Wilcox

    Analytical Biochemistry
    |May 15, 1985
    PubMed
    Summary
    This summary is machine-generated.

    This study simplifies Hong's DNA sequencing strategy, increasing yield and reducing background noise. The modified method efficiently generates sequencing subclones, improving DNA analysis.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • Hong's systematic DNA sequencing strategy offers a method for generating sequencing subclones.
    • The original protocol involved multiple steps, including gel separation, which could be time-consuming and reduce efficiency.

    Purpose of the Study:

    • To simplify Hong's DNA sequencing strategy.
    • To increase the yield of transfectants for DNA sequencing.
    • To decrease background levels in DNA sequencing protocols.

    Main Methods:

    • Modification of Hong's DNA sequencing strategy.
    • Limited DNA cleavage using DNase I in the presence of Mn2+.
    • Ligation and digestion with a second restriction endonuclease.
    • Testing the strategy on a 1.4-kb DNA fragment of the Erwinia carotovora araC gene.

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    Main Results:

    • Simplified procedure eliminates the need for gel separation of DNA.
    • Increased transfectant yield by at least 60-fold compared to the original protocol.
    • Decreased background to undetectable levels.
    • Successfully generated a set of subclones for sequencing a 1.4-kb DNA fragment in 2 days.

    Conclusions:

    • The modified DNA sequencing strategy is more efficient and simpler than the original.
    • This improved method facilitates high-yield production of sequencing subclones.
    • The strategy is effective for sequencing DNA fragments, including regulatory genes.