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PCR - Polymerase Chain Reaction01:32

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Sanger Sequencing01:57

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DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...

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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
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Novel method for PIK3CA mutation analysis: locked nucleic acid--PCR sequencing.

Daphne Ang1, Rebecca O'Gara, Amy Schilling

  • 1Department of Pathology, Oregon Health & Science University, Portland, OR, USA.

The Journal of Molecular Diagnostics : JMD
|April 2, 2013
PubMed
Summary

A novel locked nucleic acid (LNA)-PCR sequencing assay enhances PIK3CA mutation detection in breast cancer. This method shows higher sensitivity than Sanger sequencing and avoids pseudogene amplification.

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Area of Science:

  • Oncology
  • Molecular Biology
  • Genetics

Background:

  • Somatic mutations in PIK3CA are prevalent in invasive breast cancer and other carcinomas.
  • These mutations frequently occur at hotspots in exons 9 and 20.
  • Accurate detection of PIK3CA mutations is crucial for diagnosis and treatment.

Purpose of the Study:

  • To develop and validate a sensitive locked nucleic acid (LNA)-PCR sequencing assay for PIK3CA mutation detection.
  • To improve upon existing methods by avoiding amplification of a homologous PIK3CA pseudogene.
  • To assess the assay's performance in detecting low-level PIK3CA mutations in formalin-fixed paraffin-embedded (FFPE) breast DNA samples.

Main Methods:

  • Design of a locked nucleic acid (LNA)-PCR sequencing assay targeting PIK3CA hotspots (codons 542, 545, 1047).
  • Use of LNA-oligonucleotides to suppress wild-type sequence amplification.
  • Testing of 60 FFPE breast DNA samples with known PIK3CA mutation status, comparing LNA-PCR to PCR-mass spectrometry and Sanger sequencing.

Main Results:

  • The LNA-PCR assay confirmed all 51 PIK3CA mutations, achieving a 100% detection rate.
  • Standard Sanger sequencing identified only 69% of the mutations.
  • The LNA-PCR detected additional mutations in apparently normal breast tissue and hyperplasia, suggesting early mutational events.

Conclusions:

  • The developed LNA-PCR sequencing assay offers superior sensitivity for PIK3CA mutation detection compared to standard Sanger sequencing.
  • The assay effectively avoids amplification of the PIK3CA pseudogene.
  • This sensitive method has implications for understanding the role of PIK3CA mutations in various breast tissue types.