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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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PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

Katharina Wimmer¹, Annekatrin Wernstedt

¹Division of Human Genetics, Medical University Innsbruck, Peter-Mayr-Strasse 1, Innsbruck, 6020, Austria, katharina.wimmer@i-med.ac.at.

Methods in Molecular Biology (Clifton, N.J.)

|May 15, 2014

Summary

Mutation analysis of the PMS2 gene is challenging due to its pseudogene, PMS2CL. Direct cDNA sequencing effectively distinguishes between PMS2 and PMS2CL, enabling accurate detection of various mutations.

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Area of Science:

Genetics
Molecular Biology
Bioinformatics

Background:

Highly homologous pseudogenes can interfere with accurate gene mutation analysis, particularly when using PCR-based methods.
The PMS2 gene and its pseudogene, PMS2CL, share homologous sequences, complicating differentiation based on standard reference sequences.
Functional PMS2 alleles can contain pseudogene-derived sequences, and nonfunctional PMS2CL alleles can contain gene-derived sequences, rendering standard mutation detection difficult.

Purpose of the Study:

To address the challenge of distinguishing the PMS2 gene from its highly homologous pseudogene, PMS2CL.
To develop and validate a method for accurate mutation analysis of the PMS2 gene, overcoming limitations of DNA-based approaches.
To improve the detection of various genetic alterations, including deletions, splice mutations, and retrotransposon insertions.

Main Methods:

Utilized direct cDNA sequencing based on the selective amplification of PMS2 transcripts.
Employed two overlapping 1.6-kb reverse transcription PCR (RT-PCR) products for comprehensive transcript coverage.
Focused on preventing pseudogene co-amplification and allele dropout during the analysis.

Main Results:

Successfully circumvented the limitations of distinguishing PMS2 from PMS2CL by using direct cDNA sequencing.
Achieved selective amplification of PMS2 transcripts, avoiding co-amplification of the PMS2CL pseudogene.
Demonstrated the method's capability to identify deletions, splice mutations, and de novo retrotransposon insertions.

Conclusions:

Direct cDNA sequencing is a robust method for accurate mutation analysis of the PMS2 gene in the presence of homologous pseudogenes.
This approach overcomes the challenges posed by sequence exchange between PMS2 and PMS2CL, enabling reliable genetic analysis.
The described RT-PCR strategy enhances mutation detection sensitivity, identifying alterations missed by conventional DNA-based methods.