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Biomedical subjects

I G Gut

Publications and source records attributed to I G Gut.

9 recordsLinked to original sources
PubMed2001

Automation in genotyping of single nucleotide polymorphisms.

Automation for genotyping of single nucleotide polymorphisms (SNPs) can be split into the automation of the sample preparation and the automation of the analysis technology. SNP genotyping methods are reviewed and solutions for their automation discussed. A panacea for SNP genotyping does not exist. Different scientific questions require adapted solutions. The choice of a technology for SNP genotyping depends on whether few different SNPs are to be genotyped in many individuals, or many different SNPs are to be genotyped in few individuals. The requirements of throughput and the ease of establishing an SNP genotyping operation are important, as well as the degree of integration. The potential and state-of-the-art of different solutions are outlined.

Genotype↗
PubMed2000

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay.

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of ss-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the ss-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.

Animals↗
PubMed2000

A novel procedure for efficient genotyping of single nucleotide polymorphisms.

Due to the surge in interest in using single nucleotide polymorphisms (SNPs) for genotyping a facile and affordable method for this is an absolute necessity. Here we introduce a procedure that combines an easily automatable single tube sample preparation with an efficient high throughput mass spectrometric analysis technique. Known point mutations or single nucleotide polymorphisms are easily analysed by this procedure. It starts with PCR amplification of a short stretch of genomic DNA, for example an exon of a gene containing a SNP. By shrimp alkaline phosphatase digest residual dNTPs are destroyed. Allele-specific products are generated using a special primer, a conditioned set of alpha-S-dNTPs and alpha-S-ddNTPs and a fresh DNA polymerase in a primer extension reaction. Unmodified DNA is removed by 5'-phospho-diesterase digestion and the modified products are alkylated to increase the detection sensitivity in the mass spectrometric analysis. All steps of the preparation are simple additions of solutions and incubations. The procedure operates at the lowest practical sample volumes and in contrast to other genotyping protocols with mass spectrometric detection requires no purification. This reduces the cost and makes it easy to implement. Here it is demonstrated in a version using positive ion detection on described mutations in exon 17 of the amyloid precursor protein gene and in a version using negative ion detection on three SNPs of the granulocyte-macrophage colony stimulating factor gene. Preparation and analysis of SNPs is shown separately and simultaneously, thus demonstrating the multiplexibility of this genotyping procedure. The preparation protocol for genotyping is adapted to the conditions used for the SNP discovery method by denaturing HPLC, thus demonstrating a facile link between protocols for SNP discovery and SNP genotyping. Results corresponded unanimously with the control sequencing. The procedure is useful for high throughput genotyping as it is required for gene identification and pharmacogenomics where large numbers of DNA samples have to be analysed. We have named this procedure the 'GOOD Assay' for SNP analysis.

Amyloid beta-Protein Precursor↗
PubMed1999

Analysis of negatively 'charge tagged' DNA by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

An improvement in detectability and stability of DNA analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) using oligonucleotides modified with a neutralized backbone and a fixed single, positive charge was recently reported. The attachment of the positive charge requires a primary amino group, limiting this approach to accordingly functionalized DNA. The method described here uses backbone neutralized DNA for the same purpose, with a single unmodified phosphate in the DNA backbone carrying the negative charge. Thus, no chemical modification other than neutralizing the remaining charges on the phosphorothioates is required. This is performed in a single methylation step. The enhancement in sensitivity is comparable to that for DNA carrying a single positive charge, interestingly even when using the same non-protonating matrix. The mechanistic implications of these findings regarding the MALDI process are discussed. The DNA derivatization methods presented help to make MALDI-MS of DNA applicable and competitive for genome analysis and medical diagnostics.

DNA↗
PubMed1996

Evolutionary dynamics of non-coding sequences within the class II region of the human MHC.

About 40% (350 kb) of the human MHC class II region has been sequenced and a coordinated effort to sequence the entire MHC is underway. In addition to the coding information (22 genes/pseudogenes), the non-coding sequences reveal novel information on the organisation and evolution of the MHC as demonstrated here by the example of a 200 kb contig that has been analysed for local and global features. In conjunction with cross-species comparisons, our results present new evidence on the structure of isochores, the evolutionary dynamics of repeat-mediated recombination and its effect on certain MHC encoded genes, and a higher than average degree of natural polymorphism that has implications for sequencing the human genome. We also report the finding of a class I-related pseudogene (HLA-ZI) in the middle of the class II region, which provides the first direct evidence for DNA exchange between these two related regions in man.

Amino Acid Sequence↗
PubMed1996

DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Animals↗
PubMed1995

A procedure for selective DNA alkylation and detection by mass spectrometry.

A method which improves the detectability of DNA by mass spectrometry is presented. By quantitatively alkylating the backbone of phosphorothioate oligonucleotides the problems of gas phase ion generation by matrix assisted laser desorption ionization can be controlled. We have developed a selective alkylating protocol for phosphorothioate oligonucleotides which is a facile way of generating non-ionic nucleic acids. A variety of alkylating agents was studied and their kinetics were monitored in a gel electrophoretic assay and by mass spectrometry.

Alkylation↗
PubMed1993

Upper excited state photochemistry of DNA.

The quantum yields for cyclobutylpyrimidine dimers, alkali-labile sites, and frank strand breaks in double-stranded DNA have been measured using low-intensity radiation at 199.8, 217.8, and 239.5 nm from a Raman-shifted frequency quadrupled Nd:YAG laser. The quantum yield for cyclobutylpyrimidine dimers was also measured using 254 nm radiation from a low-pressure mercury lamp. The quantum yield for cyclobutylpyrimidine dimers is constant within a factor of two between 254 and 199.8 nm except for 239.5 nm, indicating that upper excited singlet states of bases convert efficiently to the lowest singlet state. The quantum yields for alkali-labile sites and frank strand breaks both increase as the wavelength decreases but follow different patterns. These results indicate that alkali-labile sites from a higher excited state of the base, whereas frank strand breaks form by excitation of the sugar-phosphate backbone.

DNA↗