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M Jobs

Publications and source records attributed to M Jobs.

5 recordsLinked to original sources
PubMed2004

DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications.

Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se)-a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses.

Algorithms↗
PubMed2001

SNP association studies in Alzheimer's disease highlight problems for complex disease analysis.

Genetic linkage and association analyses are two distinct approaches to understanding the genetic etiology of complex disease. Association analysis has become particularly popular in recent times, but the true utility of the strategy remains uncertain. To try to gain better insight into the relevant issues, we have used genetic association analysis to explore the etiology of Alzheimer's disease. Our empirical findings supplement the theoretical debate, illustrating the general doubtfulness of previous positive findings and the limited ability of typical association studies based on candidate genes to discern true medium-sized signals from false positives. Improvements in genotyping technologies and increasing the number of SNPs tested, without sophisticated allowance for all other issues, could simply lead to an unmanageable overload of false-positive signals, themselves obscuring true disease associations.

Alzheimer Disease↗
PubMed2001

Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

Alleles↗
PubMed1999

Identification of 167 polymorphisms in 88 genes from candidate neurodegeneration pathways.

Catalogs of intra-gene polymorphisms are needed to facilitate wide-ranging candidate gene-based association studies in common complex diseases. With this in mind, we have scanned multiple alignments of expressed sequence tags and of genomic DNA sequences (PCR products from four to eight unrelated individuals) to find polymorphisms in 195 genes putatively involved in neurodegenerative illness (including components of oxidative stress, excitotoxicity, inflammation, apoptosis and aging). This led to the discovery of 167 polymorphisms in 88 genes. These comprised 163 single nucleotide polymorphisms, one insertion/deletion, and three other variations involving more than one base pair. The polymorphisms were distributed in the exons (87), introns (70), and gene flanking regions (10). Of the exonic polymorphisms, 17 would give rise to non-synonymous amino acid substitutions. These findings now provide a valuable resource for association studies in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease.

Evolution, Molecular↗