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Re-sequencing of multiple single nucleotide polymorphisms by liquid chromatography-electrospray ionization mass
H Oberacher1, P J Oefner, G Hölzl
1Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens-University, Innrain 52a, A-6020 Innsbruck, Austria.
Nucleic Acids Research
|July 24, 2002
Summary
This study presents a novel method for genetic analysis, enabling precise allelic discrimination and phase determination of multiple genetic variations. The technique utilizes advanced chromatography and mass spectrometry for accurate genotyping with minimal sample amounts.
Area of Science:
- Genetics and Genomics
- Analytical Chemistry
- Biotechnology
Background:
- Accurate allelic discrimination and phase determination of genetic variations are crucial in genetics.
- Existing methods may have limitations in throughput or sensitivity for complex genotyping.
Purpose of the Study:
- To demonstrate a novel method for simultaneous genotyping of multiple polymorphisms.
- To determine the allelic phase of variations using advanced analytical techniques.
Main Methods:
- Physicochemical separation of alleles using ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC).
- On-line sequence determination by electrospray ionization mass spectrometry (ESI-MS).
- Utilizing gas-phase fragmentation and tandem mass spectrometry (MS/MS) for comparative sequencing.
Main Results:
- Simultaneous genotyping of two and three simple sequence polymorphisms (SSPs) in 73-114 bp fragments was achieved.
- Low femtomolar amounts of unpurified polymerase chain reaction (PCR) amplicons were sufficient for analysis.
- High accuracy (better than 0.019%) in intact mass measurements enabled allelic composition determination.
Conclusions:
- The combined IP-RP-HPLC and ESI-MS approach provides a powerful tool for genetic analysis.
- This method allows for accurate and efficient genotyping, including phase determination, with minimal sample requirements.
- Automated data interpretation facilitates the application of this technique in various genetic studies.