We present a novel methodology to determine the phase of single-nucleotide polymorphisms (SNPs) on a chromosome, which we term clone-based systematic haplotyping (CSH). The CSH procedure is based on separating the allelic chromosomes of a diploid genome by fosmid/cosmid cloning, and subsequent SNP typing of 96 clone pools, each representing ∼10% of the genome. The pools are screened by PCR for the sequence of interest, followed by SNP typing on the PCR products using the GOOD assay. We demonstrate that by CSH, the haplotype of SNPs separated by more than 50 kilobases can definitely be assigned. We propose this method as being suitable for constructing maps of ancestral haplotypes, analysis of complex diseases, and for diagnosis of rare defects in which the molecular haplotype is crucial. In addition, by amplifying the initial DNA by many orders of magnitude, the original DNA resource is effectively immortalized, enabling the haplotyping of hundreds of thousands of SNPs per individual.
In this article, we describe the application of a novel micro elution solid-phase extraction method for purification of short stretches of DNA products of single nucleotide polymorphisms (SNPs) prior to MALDI mass spectrometry analysis. An important feature of our method is that the purification columns containing a copolymer of divinylbenzene and N-vinylpyrrolidone can be used several times thereby significantly reducing costs. We implemented this DNA purification technology into a fully automated procedure including molecular biology, MALDI sample preparation, automated mass spectrometric analysis and SNP allele calling by software. Due to the facile purification protocol, the methodology shown could furthermore be used for other applications where efficient medium to high-throughput purification of nucleic acids is required.
Background: Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is a powerful tool in biomolecule analysis with a wide range of application possibilities, including genotyping of singlebase variations (also known as single-nucleotide polymorphisms, or SNPs) for candidate gene studies and diagnostic typing of DNA markers. We tested a method that does not require stringent purification of the nucleic acids and/or the use of modification chemistry before mass spectrometry analysis. Methods: We used an alternative direct analysis approach that allows MALDI analysis of crude DNA samples printed on microscope slides densely coated with primary amino groups that efficiently bind negatively charged DNA. After simple washing of the slides, we applied MALDI matrix and used a conventional MALDI mass spectrometer to detect DNA products. Results: We analyzed crude oligonucleotide samples and performed automated genotyping of single-base variations in 72 DNA samples. Conclusion: This procedure offers an operational short-cut compared with standard MALDI procedures for preparation of oligonucleotides, including purification, and thus is an efficient tool for genotyping applications, particularly those requiring accurate, flexible, and rapid data generation and medium throughput.
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