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Related Concept Videos

Genetic Screens02:46

Genetic Screens

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Genetic screens are tools used to identify genes and mutations responsible for phenotypes of interest. Genetic screens help identify individuals or a group of people at risk of developing  genetic diseases and help them with early intervention, targeted therapy, and reproductive options.
Forward genetic screens
Forward or “classical” genetic screens involve creating random mutations in an organism’s DNA using radiation, mutagens, or insertion of additional bases, which...
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Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
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A sensitive mutation screening method supporting cell line development for biotherapeutics.

Ildana Valisheva1, Reed J Harris2, Judith Zhu-Shimoni1

  • 1Protein Analytical Chemistry Department, Genentech, South San Francisco, CA 94080, USA.

Analytical Biochemistry
|April 25, 2016
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Summary

Detecting low-level genetic mutations in cell lines is crucial for pharmaceutical quality. A new polymerase chain reaction (PCR) and high-resolution melting (HRM) method effectively screens for these sequence variants.

Keywords:
EnrichmentHigh-resolution melt (HRM)Limiting dilutionMutationNucleic acidSequence variant (SV)

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Pharmaceutical Science

Background:

  • Cell line development for recombinant pharmaceuticals can introduce random genetic mutations.
  • These sequence variants can negatively impact the quality of the final pharmaceutical product.
  • Effective mutation screening is essential to ensure clone selection and product integrity.

Purpose of the Study:

  • To develop and validate a sensitive method for detecting low-level genetic mutations in cell lines.
  • To improve the quality control process during recombinant pharmaceutical production.
  • To identify sequence variants early in cell line development.

Main Methods:

  • A polymerase chain reaction (PCR)-based mutation screening approach was employed.
  • High-resolution melting (HRM) analysis was combined with a mutation enrichment step.
  • Limiting dilution was used to enrich for samples with low-level mutations (detectable down to 0.5%).

Main Results:

  • The developed method successfully detected low-level mutations.
  • The approach enabled the discovery of unknown mutations irrespective of their location within the transgene.
  • Mutation detection was independent of the mutation's position within the high-resolution melting (HRM) fragment (200–300 bp).

Conclusions:

  • The integrated PCR-HRM and limiting dilution method offers a robust strategy for sensitive mutation screening.
  • This technique enhances the quality control of cell lines used in pharmaceutical production.
  • Early detection of sequence variants using this method minimizes risks associated with pharmaceutical product quality.