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Mutation detection by capillary denaturing high-performance liquid chromatography using monolithic columns.

C G Huber1, A Premstaller, W Xiao

  • 1Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, A-6020, Innsbruck, Austria. christian.huber@uibk.ac.at

Journal of Biochemical and Biophysical Methods
|February 17, 2001
PubMed
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This study demonstrates mutation screening using ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) in monolithic capillary columns. This method effectively identifies single nucleotide substitutions in nucleic acids for genetic analysis.

Area of Science:

  • Molecular Biology
  • Analytical Chemistry
  • Genetics

Background:

  • High-performance liquid chromatography (HPLC) offers advanced separation capabilities for nucleic acids.
  • Mutation screening is crucial for genetic analysis and disease diagnosis.
  • Monolithic capillary columns provide high resolving power for complex biological samples.

Purpose of the Study:

  • To utilize the high resolving power of monolithic capillary columns for mutation screening.
  • To develop a method for identifying single nucleotide substitutions in polymerase chain reaction (PCR) amplified polymorphic loci.
  • To assess the efficacy of ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) in mutation detection.

Main Methods:

  • Employing 200 microm i.d. monolithic poly(styrene-divinylbenzene) capillary columns for nucleic acid separation.

Related Experiment Videos

  • Utilizing ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) under partially denaturing conditions.
  • Separating homo- and heteroduplex species to recognize mutations based on characteristic peak patterns.

    • Main Results:

    • Successfully identified six different single nucleotide substitutions in 413 bp amplicons from heterozygous individuals.
    • Each heterozygous individual exhibited a unique chromatographic profile, confirming mutation detection.
    • Resolved single-stranded nucleic acids of identical length differing by a single nucleotide in short PCR products.

    Conclusions:

    • Monolithic capillary IP-RP-HPLC is a powerful tool for mutation screening.
    • The method allows for confident identification of single nucleotide substitutions.
    • Hyphenation with mass spectrometry and array operation promise increased sample throughput for genetic analysis.