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Related Experiment Videos

Oligonucleotide-directed mutagenesis: a sequence-based screening.

A Duilio1, F Cimino, T Russo

  • 1Istituto di Scienze Biochimiche, II Facoltà di Medicina e Chirurgia, Università degli Studi di Napoli, Naples, Italy.

Analytical Biochemistry
|November 1, 1988
PubMed
Summary
This summary is machine-generated.

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A new method efficiently screens for mutant clones after mutagenesis. This technique pools phage DNA, enabling rapid identification of mutated sequences within M13 plaques using DNA sequencing.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Oligonucleotide-directed mutagenesis is crucial for genetic studies.
  • Screening for desired mutations can be time-consuming and labor-intensive.
  • Efficient methods are needed to identify mutant clones rapidly.

Purpose of the Study:

  • To develop a streamlined procedure for screening mutant clones generated by oligonucleotide-directed mutagenesis.
  • To improve the efficiency of identifying specific mutations within large populations of M13 phage.

Main Methods:

  • Preparation of phage-containing supernatants from numerous mutagenized M13 plaques.
  • Pooling of phage supernatants to create groups of approximately 10 phages.
  • Preparation of heterogeneous single-stranded DNA from pooled samples.

Related Experiment Videos

  • Application of the dideoxy chain terminator sequencing method to pooled DNA.

    • Main Results:

    • Successful development of a procedure to screen for mutant clones.
    • Identification of pools containing mutated sequences through DNA sequencing.
    • Pinpointing of the exact mutated sequence within the selected pool of 10 phages.

    Conclusions:

    • The developed procedure offers an effective method for screening mutant clones.
    • This technique significantly enhances the identification process for specific mutations.
    • The method facilitates faster genetic analysis and manipulation.