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PubMed1999

Re-sequencing of DNA from a diverse panel of cattle reveals a high level of polymorphism in both intron and exon.

In order to assess the extent of DNA sequence variation in cattle, introns and exons from both the leptin and Amyloid Precursor Protein (APP) genes have been sequenced in a panel of DNAs derived from 22 diverse animals. Direct DNA sequencing of PCR products was used; thus, 44 chromosomes were studied. Polymorphisms were identified by manual scanning of sequence chromatograms and computerized sequence analysis. Twenty Single Nucleotide Polymorphisms (SNPs) were detected in 1788 bp sequenced from the leptin gene, giving a frequency of 1 SNP per 89 bp. Twenty-four SNPs were detected in a 458-bp fragment of the APP gene; 23 of the polymorphisms were contained in a 302-bp intron 16 fragment. This equates to an SNP frequency of 1 per 13 bp for the intron. We can thus conclude that this portion of the bovine APP gene constitutes a hypermutable region. Nucleotide sequence diversity values of 0.019 and 0.0026 were obtained for APP and leptin respectively.

Amino Acid Substitution↗
PubMed2025

A vision of how low-coverage sequence data should contribute to genetic evaluation in the future.

Low-coverage sequencing refers to sequencing DNA of individuals to a low depth of coverage (e.g., 0.5X) and imputing that sequence to a genomic sequence based on reference haplotypes from individuals sequenced to a high depth of coverage (e.g., ≥10X). It has been proposed as an alternative to genotyping by Single-nucleotide polymorphisms (SNP) arrays. At least one commercial product based on it is available for agricultural species. Concerns limiting adoption in its current form are: 1) the cost of storing the huge volume of data it generates and 2) whether that additional data will result in improved accuracy of genetic evaluation. This work envisions future implementation of low-coverage sequencing to reduce storage costs and enhance genetic evaluations by leveraging the additional information in the full sequence of the pangenome to account for more genetic variation. We propose addressing the storage issue by representing genomic sequence of an individual in a pair of haplotype arrays with each element pointing to an enumerated haplotype of the sequence within one of approximately 50,000 defined genome segments. Assuming 60 million genomic variants, the infrastructure required to translate the identifier of any enumerated haplotype into its genomic sequence would require less than 10 gigabytes of binary storage. Each haplotype array element would require 2 bytes, so the marginal binary storage required to represent the genomic sequence of an individual would be about 200 kilobytes (KB), similar to the genotypes from a SNP array with 200,000 markers. This assumes no pedigree and no ambiguity of the imputation, though the latter is unrealistic. Strategies to minimize, and when necessary, to manage and efficiently represent ambiguity are proposed. The genomic sequence of an individual could be stored in about 1 KB (binary) if both parents have unambiguous sequences stored as described above. The proposed system for representing the pangenome includes algorithms for read mapping and imputation intended to leverage all known genetic variation in the target population. It is also designed to use sequencing reads generated for imputing the genomic sequence of new individuals to identify unrecognized mutations, crossovers, and structural variants, thus continuously improving the genome representation, especially if widespread use of low-coverage sequencing in livestock industries is realized. This could make improved genetic merit and management of livestock feasible without computational burden.

Animals↗
PubMed2005

[Large-scale genotyping in research into autism spectrum disorders and attention deficit hyperactivity disorder].

INTRODUCTION AND DEVELOPMENT: Autism spectrum disorder (ASD) and attention deficit hyperactivity disorder (ADHD) are two neuropsychiatric disorders beginning in childhood that present a high degree of familial aggregation. ASD is characterised by social interaction and communication disorders, whereas patients with ADHD display persistent inattention and/or hyperactive-impulsive behaviour. With the exception of a few cases of autism in which cytogenetic anomalies or mutations have been reported in specific genes, the aetiology of these diseases remains unknown. This is a group of multifactorial diseases with several genes having a lesser effect and there is also an environmental component. Genetic linkage studies have pointed to about 20 chromosomal regions that could well contain genes that grant susceptibility to autism, to ADHD or to both disorders. The challenge to researchers lies in the clinical characterisation, recruitment of patients with ASD and ADHD, gene dosage quantification studies, comparative genomic methylation and hybridisation in order to identify chromosomal rearrangements in patients with autism and severe mental retardation. CONCLUSIONS: Genotyping large SNP-type collections that are potentially functional in genes that are candidates for these disorders, based on pharmacological, biochemical and neuropathological data together with that coming from animal models and linkage studies in a wide collection of samples from patients and controls, will enable us to identify the genetic components of these pathologies and to define their biological foundations.

Attention Deficit Disorder with Hyperactivity↗
PubMed2006

Mapping of hereditary mixed polyposis syndrome (HMPS) to chromosome 10q23 by genomewide high-density single nucleotide polymorphism (SNP) scan and identification of BMPR1A loss of function.

BACKGROUND: Hereditary mixed polyposis syndrome (HMPS) is characterised by colonic polyps of mixed histological types that are autosomal dominantly inherited and eventually lead to colorectal cancer (CRC). Study of the molecular basis of HMPS will enhance our knowledge of the genetic basis of the mixed polyposis-carcinoma sequence in both hereditary and sporadic CRC. METHODS/RESULTS: We performed a genomewide linkage search on 15 members of a three-generation HMPS family using the GeneChip Human Mapping 10K Array and identified a 7 cM putative linkage interval on chromosome 10q23. Subsequently, 32 members from two HMPS families were typed with nine microsatellite markers spanning the region and the linkage was confirmed with a maximum multi-point logarithm of the odds (LOD) score of 4.6 (p<0.001). The 10q23.1-10q23.31 haplotypes segregate with the disease in both families. We screened for mutations in four candidate genes within the linkage region and identified an 11 bp deletion in the bone morphogenesis protein receptor 1A (BMPR1A) gene in one family. CONCLUSIONS: Our results indicate that BMPR1A mutation accounts for HMPS. The data suggest that inactivating BMPR1A can initiate colorectal tumourigenesis via the mixed polyposis-carcinoma sequence.

Adenomatous Polyposis Coli↗
PubMed2003

A common IL-13 Arg130Gln single nucleotide polymorphism among Chinese atopy patients with allergic rhinitis.

Allergic rhinitis is a major public health problem and has seen its prevalence increase during the past few decades. Interleukin 13 (IL-13) has been implicated in the pathogenesis and in the regulation of immunoglobulin E (IgE) production. Single nucleotide polymorphisms (SNPs) have been found in both the coding sequence and the promoter region of IL-13, and such SNPs have been associated with allergic asthma. We have investigated whether IL-13 SNPs are associated with allergic rhinitis. Among 188 Chinese adult patients with allergic rhinitis and 87 normal controls, no significant difference was found in either allele or haplotype frequency of the SNPs between the two groups. Within patients, there was a significant association of the IL-13 Arg130Gln SNP, but not of the IL-13 promoter -1112(C/T) SNP, with serum total IgE levels. Patients with a Gln/Gln genotype showed much higher serum total IgE than those with an Arg/Arg genotype. When tested for serum-specific IgE, patients allergic to Derp 1, but not those allergic to Artemisia pollen, showed a significant association with the IL-13 promoter SNP. Thus, our results suggest a possible involvement of IL-13 SNPs in the regulation of IgE production in response to allergens in this Chinese population.

Adult↗
PubMed2003

A promoter SNP (-1323T>C) in G-substrate gene (GSBS) correlates with hypercholesterolemia.

Factors predisposing to the phenotypic features of higher total cholesterol (TC) have not been clearly defined. Here we report an association between a promoter SNP (-1323T>C) in G-substrate gene (GSBS) and TC levels in 368 adult individuals from an east-central area of Japan. Age and gender-adjusted levels of LDL-cholesterol, TG, TC, and HDL-cholesterol were analyzed. When we separate the subjects into two genotypic groups regarding T allele, those who bear the T allele had significantly higher plasma TC levels than the others who lack the T allele (mean; 239.6 mg/dl vs. 210.6 mg/dl; p=0.003; Mann-Whitney test). Of the 341 individuals with the T allele, approximately 80% individuals presented with hypercholesterolemia, whereas only 44% were hypercholesterolemic among the 27 individuals without the T allele (p=0.0001). These results indicate a significant elevating effect of plasma TC levels by a SNP in the putative regulatory region of the G-substrate gene in our studied population. These data suggest that genetic variation at the G-substrate gene may be one of the determinants for plasma lipoprotein levels.

Cholesterol↗
PubMed2006

MTHFR C677T and A1298C gene polymorphisms and hyperhomocysteinemia as risk factors of diabetic nephropathy in type 2 diabetes patients.

Point mutations in methylenetetrahydrofolate reductase (MTHFR) and hyperhomocysteinemia were implicated in the pathogenesis of diabetic nephropathy (DN) in many ethnic groups. This study addressed the association of C677T and A1298C single nucleotide polymorphisms (SNPs) of MTHFR gene with DN in Tunisian type 2 diabetes (T2DM) patients. Study subjects comprised 93 DN patients, 267 patients with normoalbuminuria, and 400 control subjects. C677T and A1298C genotypes were determined by PCR-RFLP analysis, and homocysteine levels were measured by ELISA. A1298C and C677T were highly prevalent among T2DM patients, with allele frequencies of 0.26 and 0.36, respectively. Higher mutant 677T allele and 677C/T and 677T/T genotypes of C677T SNP, but not A1298C SNP, together with 677C/1298A, 677C/1298C, and 677T/1298A haplotypes were seen in DN patients compared to normoalbuminuric patients, (p<0.001). Plasma homocysteine was positively associated with MTHFR 677T/T genotype among the three groups, and was significantly elevated in double heterozygous DN patients but not in normoalbuminuric patients or controls. Logistic regression analysis with DN as dependent variable showed that homocysteine (OR, 1.153) and MTHFR 677T/T (OR, 9.799) were the only variables associated with DN, after adjusting for possible confounding variables. C677T, but not A1298C, SNP, is a risk factor for DN, presumably acting by elevating homocysteine levels.

Adult↗
PubMed2000

A new MALDI-TOF based mini-sequencing assay for genotyping of SNPS.

A new MALDI-TOF based mini-sequencing assay termed VSET was developed for genotyping of SNPs. In this assay, specific fragments of genomic DNA containing the SNP site(s) are first amplified, followed by mini-sequencing in the presence of three ddNTPs and the fourth nucleotide in the deoxy form. In this way, the primer is extended by only one base from one allele, while it is typically extended by two bases from another allele. The products are then analyzed using MALDI-TOF mass spectrometry. The genotype of the SNP site is identified based on the number of nucleotides added. This assay has been examined using both synthetic and genomic DNA samples. In addition, multiplexed assays were successfully performed to genotype four SNP sites in a single tube. The main aspect of this assay is that it can overcome the key problems associated with the currently used mini-sequencing methods. First, it significantly reduces the stringent high-resolution and extensive desalting requirements that are essential to the pinpoint assay. Second, it avoids the long extension problem associated with the PROBE assay.

DNA Mutational Analysis↗
PubMed2005

SNP array-based homozygosity mapping reveals MCPH1 deletion in family with autosomal recessive mental retardation and mild microcephaly.

Very little is known about the molecular basis of autosomal recessive MR (ARMR) because in developed countries, small family sizes preclude mapping and identification of the relevant gene defects. We therefore chose to investigate genetic causes of ARMR in large consanguineous Iranian families. This study reports on a family with six mentally retarded members. Array-based homozygosity mapping and high-resolution microarray-based comparative genomic hybridization (array CGH) revealed a deletion of approximately 150-200 kb, encompassing the promoter and the first six exons of the MCPH1 gene, one out of four genes that have been previously implicated in ARMR with microcephaly. Reexamination of affected individuals revealed a high proportion of prematurely condensed chromosomes, which is a hallmark of this condition, but in spite of the severity of the mutation, all patients showed only borderline to mild microcephaly. Therefore the phenotypic spectrum of MCPH1 mutations may be wider than previously assumed, with ARMR being the only consistent clinical finding.

Adolescent↗
PubMed2005

In silico whole-genome scanning of cancer-associated nonsynonymous SNPs and molecular characterization of a dynein light chain tumour variant.

Last decade has led to the accumulation of large amounts of data on cancer genetics, opening an unprecedented access to the mapping of cancer genes in the human genome. Single-nucleotide polymorphisms (SNPs), the most common form of DNA variation in humans, emerge as an invaluable tool for cancer association studies. These genotypic markers can be used to assay how alleles of candidate genes correlate with the malignant phenotype, and may provide new clues into the genetic modifications that characterize cancer onset. In this cancer-oriented study, we detail an SNP mining strategy based on the analysis of expressed sequence tags among publicly available databases. Our whole-genome approach provides a comprehensive and unbiased description of nonsynonymous SNPs (nsSNPs) in tumoral versus normal tissues. To gain further insights into the possible relationships between genetic variation and altered phenotype, locations of a subset of nsSNPs were mapped onto protein domains known to be critical for protein function. Computational methods were also used to predict the potential impact of these cancer-associated nsSNPs on protein structure and function. We illustrate our approach through the detailed biochemical and structural characterization of a previously unknown cancer-associated mutation (G79C) affecting the 8 kDa dynein light chain (DNCL1).

Computational Biology↗
PubMed2004

Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.

BACKGROUND: High-resolution melting of PCR amplicons with the DNA dye LCGreen I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (</=50 bp). METHODS: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and beta-globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. RESULTS: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T(m)s) that differ by 0.8-1.4 degrees C. However, in approximately 16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T(m) differences between homozygotes (<0.4 degrees C). Although most of these cases can be genotyped by T(m), one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes. This approach was used for the HFE 187C>G protocol, but, as predicted from the sequence changes, was not needed for the other four clinical protocols. CONCLUSIONS: SNP genotyping by high-resolution melting analysis is simple, rapid, and inexpensive, requiring only PCR, a DNA dye, and melting instrumentation. The method is closed-tube, performed without probes or real-time PCR, and can be completed in less than 2 min after completion of PCR.

Factor V↗
PubMed2003

Genetic risk factors for cerebrovascular disease in children with sickle cell disease: design of a case-control association study and genomewide screen.

BACKGROUND: The phenotypic heterogeneity of sickle cell disease is likely the result of multiple genetic factors and their interaction with the sickle mutation. High transcranial doppler (TCD) velocities define a subgroup of children with sickle cell disease who are at increased risk for developing ischemic stroke. The genetic factors leading to the development of a high TCD velocity (i.e. cerebrovascular disease) and ultimately to stroke are not well characterized. METHODS: We have designed a case-control association study to elucidate the role of genetic polymorphisms as risk factors for cerebrovascular disease as measured by a high TCD velocity in children with sickle cell disease. The study will consist of two parts: a candidate gene study and a genomewide screen and will be performed in 230 cases and 400 controls. Cases will include 130 patients (TCD > or = 200 cm/s) randomized in the Stroke Prevention Trial in Sickle Cell Anemia (STOP) study as well as 100 other patients found to have high TCD in STOP II screening. Four hundred sickle cell disease patients with a normal TCD velocity (TCD < 170 cm/s) will be controls. The candidate gene study will involve the analysis of 28 genetic polymorphisms in 20 candidate genes. The polymorphisms include mutations in coagulation factor genes (Factor V, Prothrombin, Fibrinogen, Factor VII, Factor XIII, PAI-1), platelet activation/function (GpIIb/IIIa, GpIb IX-V, GpIa/IIa), vascular reactivity (ACE), endothelial cell function (MTHFR, thrombomodulin, VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1), inflammation (TNFalpha), lipid metabolism (Apo A1, Apo E), and cell adhesion (VCAM-1, E-Selectin, L-Selectin, P-Selectin, ICAM-1). We will perform a genomewide screen of validated single nucleotide polymorphisms (SNPs) in pooled DNA samples from 230 cases and 400 controls to study the possible association of additional polymorphisms with the high-risk phenotype. High-throughput SNP genotyping will be performed through MALDI-TOF technology using Sequenom's MassARRAY system. DISCUSSION: It is expected that this study will yield important information on genetic risk factors for the cerebrovascular disease phenotype in sickle cell disease by clarifying the role of candidate genes in the development of high TCD. The genomewide screen for a large number of SNPs may uncover the association of novel polymorphisms with cerebrovascular disease and stroke in sickle cell disease.

Adolescent↗
PubMed2006

KCNQ4: a gene for age-related hearing impairment?

Age-related hearing impairment (ARHI) is the most common sensory impairment among the elderly. It is a complex disorder influenced by genetic as well as environmental factors. SNPs in a candidate susceptibility gene, KCNQ4, were examined in two independent Caucasian populations. Two quantitative trait locus (QTL) values were investigated: Zhigh and Zlow, a measure of high and respectively low frequency hearing loss. In the first population, the statistical analysis of 23 genotyped SNPs spread across KCNQ4 resulted in significant p-values for two SNPs for Zhigh-SNP9 (NT_004511:g.11244177A > T) and SNP15 (NT_004511:g.11257005C > T; NP_004691:p.Ala259Ala), and one SNP for Zlow-SNP12 (NT_004511:g.11249550A > T). The linkage disequilibrium (LD) structure of KCNQ4 was subsequently determined in a 34-kb region surrounding the significant SNPs, resulting in three LD-blocks. LD-block 1 contains SNP9 and covers an area of 5 kb, LD-block 2 measures 5 kb and surrounds SNP13 (NT_004511:g.11253513A > G) to SNP18 (NT_004511:g.11257509G > A; NP_004691:p.Thr293Thr), and LD-block 3 spans 7 kb. Five tag-SNPs of block 1 and 2, and 2 extra SNPs were subsequently genotyped in the second population. Again, several SNPs were positively associated with ARHI: one SNP (SNP18) for the high frequencies and three SNPs (SNP9, SNP12, and SNP18) for the low frequencies, although only a single SNP (SNP12) resulted in significant p-values in both populations. Nevertheless, the associated SNPs of both populations were all located in the same 13-kb region in the middle of the KCNQ4 gene.

Adult↗
PubMed2006

Enhanced sensitivity to oxidative stress in an Arabidopsis nitric oxide synthase mutant.

The possible involvement of nitric oxide (NO) in oxidative stress tolerance was studied using Arabidopsis thaliana wild type (WT) and Atnos1 mutant plants, in which endogenous NO production is greatly diminished because 80% of nitric oxide synthase (NOS) activity is eliminated due to T-DNA insertion in the first exon of the NOS1 gene. Compared with WT, Atnos1 mutant plants showed increased hypersensitivity to salt stress and methyl viologen (MV) treatment. The maximal photochemical efficiency of photosystem II (F(v)/F(m)) and membrane integrity decreased in WT and Atnos1 mutant plants under stresses, but the extent was higher in the mutant. Treatment with sodium nitroprusside (SNP) (a NO donor) to Atnos1 mutant plants alleviated the damage. Instead, inhibition of nitric oxide accumulation in the WT plants produced opposite effects. Hydrogen peroxide and lipid peroxidation increased and the extent was higher in Atnos1 mutant plants than that in WT plants under MV stress. These results indicated that nitric oxide could protect the damage against NaCl and MV treatments.

Arabidopsis↗
PubMed2005

Mining genetic epidemiology data with Bayesian networks I: Bayesian networks and example application (plasma apoE levels).

MOTIVATION: The wealth of single nucleotide polymorphism (SNP) data within candidate genes and anticipated across the genome poses enormous analytical problems for studies of genotype-to-phenotype relationships, and modern data mining methods may be particularly well suited to meet the swelling challenges. In this paper, we introduce the method of Belief (Bayesian) networks to the domain of genotype-to-phenotype analyses and provide an example application. RESULTS: A Belief network is a graphical model of a probabilistic nature that represents a joint multivariate probability distribution and reflects conditional independences between variables. Given the data, optimal network topology can be estimated with the assistance of heuristic search algorithms and scoring criteria. Statistical significance of edge strengths can be evaluated using Bayesian methods and bootstrapping. As an example application, the method of Belief networks was applied to 20 SNPs in the apolipoprotein (apo) E gene and plasma apoE levels in a sample of 702 individuals from Jackson, MS. Plasma apoE level was the primary target variable. These analyses indicate that the edge between SNP 4075, coding for the well-known epsilon2 allele, and plasma apoE level was strong. Belief networks can effectively describe complex uncertain processes and can both learn from data and incorporate prior knowledge. AVAILABILITY: Various alternative and supplemental networks (not given in the text) as well as source code extensions, are available from the authors. SUPPLEMENTARY INFORMATION: http://bioinformatics.oxfordjournals.org.

Apolipoproteins E↗
PubMed2006

Dynamic variable selection in SNP genotype autocalling from APEX microarray data.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are DNA sequence variations, occurring when a single nucleotide--adenine (A), thymine (T), cytosine (C) or guanine (G)--is altered. Arguably, SNPs account for more than 90% of human genetic variation. Our laboratory has developed a highly redundant SNP genotyping assay consisting of multiple probes with signals from multiple channels for a single SNP, based on arrayed primer extension (APEX). This mini-sequencing method is a powerful combination of a highly parallel microarray with distinctive Sanger-based dideoxy terminator sequencing chemistry. Using this microarray platform, our current genotype calling system (known as SNP Chart) is capable of calling single SNP genotypes by manual inspection of the APEX data, which is time-consuming and exposed to user subjectivity bias. RESULTS: Using a set of 32 Coriell DNA samples plus three negative PCR controls as a training data set, we have developed a fully-automated genotyping algorithm based on simple linear discriminant analysis (LDA) using dynamic variable selection. The algorithm combines separate analyses based on the multiple probe sets to give a final posterior probability for each candidate genotype. We have tested our algorithm on a completely independent data set of 270 DNA samples, with validated genotypes, from patients admitted to the intensive care unit (ICU) of St. Paul's Hospital (plus one negative PCR control sample). Our method achieves a concordance rate of 98.9% with a 99.6% call rate for a set of 96 SNPs. By adjusting the threshold value for the final posterior probability of the called genotype, the call rate reduces to 94.9% with a higher concordance rate of 99.6%. We also reversed the two independent data sets in their training and testing roles, achieving a concordance rate up to 99.8%. CONCLUSION: The strength of this APEX chemistry-based platform is its unique redundancy having multiple probes for a single SNP. Our model-based genotype calling algorithm captures the redundancy in the system considering all the underlying probe features of a particular SNP, automatically down-weighting any 'bad data' corresponding to image artifacts on the microarray slide or failure of a specific chemistry. In this regard, our method is able to automatically select the probes which work well and reduce the effect of other so-called bad performing probes in a sample-specific manner, for any number of SNPs.

Algorithms↗
PubMed2002

Efficient and high fidelity incorporation of dye-terminators by a novel archaeal DNA polymerase mutant.

We examined the molecular basis of ddNTP selectivity in archaeal family B DNA polymerases by randomly mutagenizing the gene encoding Thermococcus sp. JDF-3 DNA polymerase and screening mutant libraries for improved ddNTP incorporation. We identified two mutations, P410L and A485T, that improved ddNTP uptake, suggesting the contribution of P410 and A485 to ddNTP/dNTP selectivity in archaeal DNA polymerases. The importance of A485 was identified previously in mutagenesis studies employing Pfu (A486) and Vent (A488) DNA polymerases, while the contribution of P410 to ddNTP/dNTP selectivity has not been reported. We demonstrate that a combination of mutations (P410L/A485T) has an additive effect in improving ddNTP incorporation by a total of 250-fold. To assess the usefulness of the JDF-3 P410L/A485T in fluorescent-sequencing applications, we compared the archaeal mutant to Taq F667Y with respect to fidelity and kinetic parameters for DNA and dye-ddNTPs. Although the Taq F667Y and JDF-3 P410L/A485T mutants exhibit similar K(m) and V(max) values for dye-ddNTPs in single-base extension assays, the archaeal mutant exhibits higher fidelity due to a reduced tendency to form certain (ddG:dT, ddT:dC) mispairs. DNA polymerases exhibiting higher insertion fidelity are expected to provide greater accuracy in SNP frequency determinations by single-base extension and in multiplex minisequencing assays.

Base Sequence↗
PubMed2005

Highly multiplexed molecular inversion probe genotyping: over 10,000 targeted SNPs genotyped in a single tube assay.

Large-scale genetic studies are highly dependent on efficient and scalable multiplex SNP assays. In this study, we report the development of Molecular Inversion Probe technology with four-color, single array detection, applied to large-scale genotyping of up to 12,000 SNPs per reaction. While generating 38,429 SNP assays using this technology in a population of 30 trios from the Centre d'Etude Polymorphisme Humain family panel as part of the International HapMap project, we established SNP conversion rates of approximately 90% with concordance rates >99.6% and completeness levels >98% for assays multiplexed up to 12,000plex levels. Furthermore, these individual metrics can be "traded off" and, by sacrificing a small fraction of the conversion rate, the accuracy can be increased to very high levels. No loss of performance is seen when scaling from 6,000plex to 12,000plex assays, strongly validating the ability of the technology to suppress cross-reactivity at high multiplex levels. The results of this study demonstrate the suitability of this technology for comprehensive association studies that use targeted SNPs in indirect linkage disequilibrium studies or that directly screen for causative mutations.

Chromosome Inversion↗