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PubMed2004

Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel.

Comprehensive genome scans involving many thousands of SNP assays will require significant amounts of genomic DNA from each sample. We report two successful methods for amplifying whole-genomic DNA prior to SNP analysis, multiple displacement amplification, and OmniPlex technology. We determined the coverage of amplification by analyzing a SNP linkage marker set that contained 2320 SNP markers spread across the genome at an average distance of 2.5 cM. We observed a concordance of >99.8% in genotyping results from genomic DNA and amplified DNA, strongly indicating the ability of both methods used to amplify genomic DNA in a highly representative manner. Furthermore, we were able to achieve a SNP call rate of >98% in both genomic and amplified DNA. The combination of whole-genome amplification and comprehensive SNP linkage analysis offers new opportunities for genetic analysis in clinical trials, disease association studies, and archiving of DNA samples.

DNA↗

PubMed1999

Vascular dilatatory responses to sodium nitroprusside (SNP) and alpha-adrenergic antagonism in female and male normal and diabetic rats.

Diabetes is associated with impaired vascular dilatatory responses that appear to be influenced by sex as well as diabetic state. Therefore, we hypothesized that vascular and sympathetic control function exhibit a greater deterioration following the induction of diabetes in female than in male rats. We conducted a comparative determination of the effect of sodium nitroprusside (SNP, a nitrous oxide donor) and that of an alpha1-adrenergic antagonist, prazosin, on selective vascular flows, mean arterial pressure (MAP), and heart rate (HR), in female and male normal and diabetic rats. Rats were made diabetic using streptozotocin (50 mg/kg, iv) and maintained for 5-6 weeks. Following anesthesia with urethane/alpha-chloralose, the femoral artery and vein were cannulated for recording and sampling. Flow probes were placed on the iliac, renal, and superior mesenteric arteries. SNP (1, 5, 10, and 20 microg/kg) infusions resulted in a dose-dependent decrease in MAP in normal and diabetic rats. The decrease in MAP in normal males was 37% less at the 20 microg/kg concentration of SNP when compared to normal females. The HR was not significantly changed in response to the hypotensive effect of SNP; however, reflex tachycardia was more prominent in diabetic males. The vascular conductance (flow/MAP) was increased by SNP in normal and diabetic rats in a dose-dependent fashion; however, the responsiveness was decreased in the iliac and superior mesenteric and increased in the renal arteries in diabetics when compared to normals. Diabetic males were 42% and 28% less responsive to SNP in the iliac and superior mesenteric arteries, respectively. On the other hand, diabetic females were 1.5-fold more responsive in the renal artery when compared to normals. Prazosin (4 mg/kg) decreased the MAP in normal and diabetic rats to a comparable degree. Prazosin increased the vascular conductance in all three vascular beds in normal and diabetic rats with the greater increase occurring in the iliac (118%) and superior mesenteric (110%) arteries. We concluded that diabetes is associated with an increased response to NO in the renal vessels and a decreased response in the iliac and superior mesenteric vessels in both females and males. alpha-Adrenergic tone was greatest in diabetic female and male rats. This study suggests that decreased vascular flow in diabetes is a result of a combination of decreased sensitivity to NO and increased adrenergic tone.

Adrenergic alpha-Antagonists↗

PubMed2005

Intracavernous sodium nitroprusside (SNP) versus papaverine/phentolamine in erectile dysfunction: a comparative study of short-term efficacy and side-effects.

OBJECTIVE: The aim of our work is to evaluate the efficacy of intracavernous sodium nitroprusside (SNP) in management of erectile dysfunction (ED) in a clinical comparative study with papaverine/phentolamine in ED patients. METHODS: The study included 40 patients with ED divided into two groups. Group I include 20 patients receiving intracavernous (30 mg papaverine + 1 mg phentolamine) followed 1 week later by intracavernous 300 microg SNP. Group II included 20 patients receiving the same regimen of group I but with intracavernous SNP first followed by papaverine/phentolamine 1 week later. All patients were assessed clinically for their response and any developing complications. RESULTS: The numbers of good and poor responders were not statistically significant (P > 0.05) among the two groups. The mean erectile duration of SNP was similar to bimix (P > 0.05). No side-effects whether local or systemic occurred with SNP while priapism and local penile pain were recorded with bimix solution. CONCLUSIONS: Intracavernous pharmacotherapy is still a reliable method both for diagnosis and for treatment of ED. While preliminary results of our study show a potential of SNP to be an effective and safe intracavernous agent, long-term self-injection clinical trials are needed before large-scale usage is recommended.

Adrenergic alpha-Antagonists↗

PubMed1997

Tyrosine phosphorylation in contraction of opossum esophageal longitudinal muscle in response to SNP.

Sodium nitroprusside (SNP) has been shown to elicit a guanosine 3',5'-cyclic monophosphate (cGMP)-mediated, indomethacin-sensitive contraction of the opossum esophageal longitudinal muscle. We examined the role of tyrosine phosphorylation in the signal transduction pathway of contractions induced by SNP and cGMP in longitudinal muscle strips in vitro. Force of isometric contractions was expressed as the percentage of responses to KCl (73 mM). SNP (100 microM)-induced contractions were 75 +/- 5% before and 3 +/- 2% after 50 microM genistein (P < 0.005) and 86 +/- 16% before and 0 +/- 0% after 50 microM tyrphostin B46. Contractions in response to 8-bromo-cGMP (8-BrcGMP; 1 mM) were 74 +/- 15% before and 3 +/- 2% after genistein (P < 0.01) and 63 +/- 15% before and 18 +/- 4% after tyrphostin B46 (P < 0.05). In contrast, KCl-induced contractions were 82 +/- 8% and 96 +/- 9% of the control value after genistein and tyrphostin B46 treatments, respectively (P > 0.05 for both). Carbachol contractions were partially suppressed by genistein (106 +/- 8% vs. 79 +/- 8%; P < 0.05) but unaffected by tyrphostin B46 (114 +/- 10% vs. 107 +/- 12%; P > 0.05). Western blot analysis revealed a 116-kDa phosphotyrosine protein in the control muscle strips. The level of this protein was increased to 206 +/- 15% of control after SNP treatment. Both genistein and tyrphostin B46 blocked this increase. These studies show that contractions of the esophageal longitudinal muscle induced by SNP and cGMP utilize a signal transduction pathway different from that used by the depolarizing agent KCl and the muscarinic agonist carbachol. Contractions induced by SNP and cGMP involve tyrosine phosphorylation of a protein, possibly identified as a 116-kDa protein, as a key step in the signaling pathway.

Animals↗

PubMed2006

The impact of missing and erroneous genotypes on tagging SNP selection and power of subsequent association tests.

OBJECTIVE: Single nucleotide polymorphisms (SNPs) serve as effective markers for localizing disease susceptibility genes, but current genotyping technologies are inadequate for genotyping all available SNP markers in a typical linkage/association study. Much attention has recently been paid to methods for selecting the minimal informative subset of SNPs in identifying haplotypes, but there has been little investigation of the effect of missing or erroneous genotypes on the performance of these SNP selection algorithms and subsequent association tests using the selected tagging SNPs. The purpose of this study is to explore the effect of missing genotype or genotyping error on tagging SNP selection and subsequent single marker and haplotype association tests using the selected tagging SNPs. METHODS: Through two sets of simulations, we evaluated the performance of three tagging SNP selection programs in the presence of missing or erroneous genotypes: Clayton's diversity based program htstep, Carlson's linkage disequilibrium (LD) based program ldSelect, and Stram's coefficient of determination based program tagsnp.exe. RESULTS: When randomly selected known loci were relabeled as 'missing', we found that the average number of tagging SNPs selected by all three algorithms changed very little and the power of subsequent single marker and haplotype association tests using the selected tagging SNPs remained close to the power of these tests in the absence of missing genotype. When random genotyping errors were introduced, we found that the average number of tagging SNPs selected by all three algorithms increased. In data sets simulated according to the haplotype frequecies in the CYP19 region, Stram's program had larger increase than Carlson's and Clayton's programs. In data sets simulated under the coalescent model, Carlson's program had the largest increase and Clayton's program had the smallest increase. In both sets of simulations, with the presence of genotyping errors, the power of the haplotype tests from all three programs decreased quickly, but there was not much reduction in power of the single marker tests. CONCLUSIONS: Missing genotypes do not seem to have much impact on tagging SNP selection and subsequent single marker and haplotype association tests. In contrast, genotyping errors could have severe impact on tagging SNP selection and haplotype tests, but not on single marker tests.

Algorithms↗

PubMed2006

Evidence for consistent intragenic and intergenic interactions between SNP effects in the APOA1/C3/A4/A5 gene cluster.

OBJECTIVE: Evaluate the consistency of the contribution of interactions between single nucleotide polymorphism (SNP) genotype effects to variation in measures of lipid metabolism across ethnic strata within gender. METHODS AND RESULTS: We considered 80 SNPs within the apolipoprotein (APO) A1/C3/A4/A5 gene cluster using an over-parameterized general linear model to identify SNPs whose genotype effects combine non-additively to influence plasma levels of high density lipoprotein cholesterol (HDL-C), total cholesterol (TC) and triglycerides (TG) in a consistent manner across ethnic strata. We analyzed population-based samples of unrelated 18 to 30 year old African-Americans (n = 1,858) and European-Americans (n = 1,973) ascertained without regard to health at four field centers (Birmingham, Ala.; Chicago, Ill.; Minneapolis, Minn. and Oakland, Calif., USA) by the Coronary Artery Risk Development in Young Adults (CARDIA) study. To identify which SNP genotype effects combine non-additively we used a two-tier analysis strategy. We first required that pairs of SNPs show statistically significant non-additivity in both ethnic strata within a gender, where experiment-wise significance was evaluated using a permutation test to determine the probability of observing the number of tests significant in both ethnic strata by chance alone. Second, we required no significant evidence of heterogeneity of the relationship between the phenotype and the two SNP genotypes across ethnic strata and across field centers within each ethnic group. From this strategy we identified ten pairs of SNPs, involving thirteen SNPs, that displayed statistically significant non-additivity of SNP genotype effects on TC. Only one of these thirteen SNPs had statistically significant genotype effects that were consistent across samples. CONCLUSION: Our analyses suggest that ignoring the contribution of interactions between SNP genotype effects when modeling multi-SNP genotype-phenotype relationships may result in an underestimate of the contribution of genetic variation to variation in quantitative cardiovascular disease (CVD) risk factor traits.

Adolescent↗

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↗

PubMed2006

Single nucleotide polymorphism (SNP) discovery in duplicated genomes: intron-primed exon-crossing (IPEC) as a strategy for avoiding amplification of duplicated loci in Atlantic salmon (Salmo salar) and other salmonid fishes.

BACKGROUND: Single nucleotide polymorphisms (SNPs) represent the most abundant type of DNA variation in the vertebrate genome, and their applications as genetic markers in numerous studies of molecular ecology and conservation of natural populations are emerging. Recent large-scale sequencing projects in several fish species have provided a vast amount of data in public databases, which can be utilized in novel SNP discovery in salmonids. However, the suggested duplicated nature of the salmonid genome may hamper SNP characterization if the primers designed in conserved gene regions amplify multiple loci. RESULTS: Here we introduce a new intron-primed exon-crossing (IPEC) method in an attempt to overcome this duplication problem, and also evaluate different priming methods for SNP discovery in Atlantic salmon (Salmo salar) and other salmonids. A total of 69 loci with differing priming strategies were screened in S. salar, and 27 of these produced approximately 13 kb of high-quality sequence data consisting of 19 SNPs or indels (one per 680 bp). The SNP frequency and the overall nucleotide diversity (3.99 x 10-4) in S. salar was lower than reported in a majority of other organisms, which may suggest a relative young population history for Atlantic salmon. A subset of primers used in cross-species analyses revealed considerable variation in the SNP frequencies and nucleotide diversities in other salmonids. CONCLUSION: Sequencing success was significantly higher with the new IPEC primers; thus the total number of loci to screen in order to identify one potential polymorphic site was six times less with this new strategy. Given that duplication may hamper SNP discovery in some species, the IPEC method reported here is an alternative way of identifying novel polymorphisms in such cases.

Animals↗

PubMed2007

A procedure for the detection of linkage with high density SNP arrays in a large pedigree with colorectal cancer.

BACKGROUND: The apparent dominant model of colorectal cancer (CRC) inheritance in several large families, without mutations in known CRC susceptibility genes, suggests the presence of so far unidentified genes with strong or moderate effect on the development of CRC. Linkage analysis could lead to identification of susceptibility genes in such families. In comparison to classical linkage analysis with multi-allelic markers, single nucleotide polymorphism (SNP) arrays have increased information content and can be processed with higher throughput. Therefore, SNP arrays can be excellent tools for linkage analysis. However, the vast number of SNPs on the SNP arrays, combined with large informative pedigrees (e.g. >35-40 bits), presents us with a computational complexity that is challenging for existing statistical packages or even exceeds their capacity. We therefore setup a procedure for linkage analysis in large pedigrees and validated the method by genotyping using SNP arrays of a colorectal cancer family with a known MLH1 germ line mutation. METHODS: Quality control of the genotype data was performed in Alohomora, Mega2 and SimWalk2, with removal of uninformative SNPs, Mendelian inconsistencies and Mendelian consistent errors, respectively. Linkage disequilibrium was measured by SNPLINK and Merlin. Parametric linkage analysis using two flanking markers was performed using MENDEL. For multipoint parametric linkage analysis and haplotype analysis, SimWalk2 was used. RESULTS: On chromosome 3, in the MLH1-region, a LOD score of 1.9 was found by parametric linkage analysis using two flanking markers. On chromosome 11 a small region with LOD 1.1 was also detected. Upon linkage disequilibrium removal, multipoint linkage analysis yielded a LOD score of 2.1 in the MLH1 region, whereas the LOD score dropped to negative values in the region on chromosome 11. Subsequent haplotype analysis in the MLH1 region perfectly matched the mutation status of the family members. CONCLUSION: We developed a workflow for linkage analysis in large families using high-density SNP arrays and validated this workflow in a family with colorectal cancer. Linkage disequilibrium has to be removed when using SNP arrays, because it can falsely inflate the LOD score. Haplotype analysis is adequate and can predict the carrier status of the family members.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Tumor classification based on DNA copy number aberrations determined using SNP arrays.

High-density single nucleotide polymorphism (SNP) array is a recently introduced technology that genotypes more than 10,000 human SNPs on a single array. It has been shown that SNP arrays can be used to determine not only SNP genotype calls, but also DNA copy number (DCN) aberrations, which are common in solid tumors. In the past, effective cancer classification has been demonstrated using microarray gene expression data, or DCN data derived from comparative genomic hybridization (CGH) arrays. However, the feasibility of cancer classification based on DCN aberrations determined by SNP arrays has not been previously investigated. In this study, we address this issue by applying state-of-the-art classification algorithms and feature selection algorithms to the DCN aberration data derived from a public SNP array dataset. Performance was measured via leave-one-out cross-validation (LOOCV) classification accuracy. Experimental results showed that the maximum accuracy was 73.33%, which is comparable to the maximum accuracy of 76.5% based on CGH-derived DCN data reported previously in the literature. These results suggest that DCN aberration data derived from SNP arrays is useful for etiology-based tumor classification.

Algorithms↗

PubMed2026

Dynamic Fusion of Genomics and Functional Network Connectivity in UK Biobank Reveals Schizophrenia-Related SNP Manifolds.

Many mental disorders show strong genetic influence. In parallel, dynamic functional network connectivity (dFNC) has shown high sensitivity to brain changes related to mental disorders. However, previous studies linking dFNC to genetics largely follow a paradigm to identify associations between one set of genetic factors and multiple sets of connectivity features from different dFNC states, ignoring the potential variability in genetic correlates across states. We propose a novel joint ICA (jICA)-based "dynamic fusion" framework to identify dynamically tuned genetic manifolds. A sliding window approach was utilized to estimate four dFNC states and compute subject-level state-average dFNC (sa-dFNC) features. The sa-dFNC features of each state were combined with schizophrenia risk single nucleotide polymorphisms (SNPs) within a jICA fusion framework, resulting in four parallel fusions in 32,861 individuals of the UK Biobank cohort. The extracted four sets of joint SNP-dFNC components were further validated for clinical relevance in a combined schizophrenia cohort of 820 individuals (348 patients). The similarity of SNP-dFNC components across four parallel fusions was evaluated as a measure of state variability. We observed a mixture of "state-invariant" and "state-variant" components for SNP and dFNC modalities. Particularly, the schizophrenia-related state-variant SNP components, or manifolds, complemented each other by capturing different SNPs involved in the same biological functions, revealing a partition of genomic risk particularly elicited by the dynamics of brain function. By augmenting the SNP factors to state-variant manifolds, this dynamic fusion framework promises additional insights into the underlying genetic risk of disease-related alterations in dynamic brain function.

Humans↗

PubMed2003

Detection of single nucleotide polymorphism (SNP) controlling the waxy character in wheat by using a derived cleaved amplified polymorphic sequence (dCAPS) marker.

We investigated a single nucleotide polymorphism (SNP) in the Wx-D1 gene, which was found in a mutant waxy wheat, and which expressed the Wx-D1 protein (granule-bound starch synthase I) as shown by immunoblot analysis. We also assayed starch synthase activity of granule-bound proteins. Using 22 doubled-haploid (DH) lines and 172 F(5) lines derived from the wild type x the mutant, we detected SNP via a PCR-based (dCAPS) marker. Amplified PCR products from Wx-D1 gene-specific primers, followed by mismatched primers designed for dCAPS analysis, were digested with the appropriate restriction enzyme. The two alleles, and the heterozygote genotype were easily and rapidly discriminated by gel-electrophoresis resolution to reveal SNP. All progeny lines that have the SNP of the mutant allele were waxy. Integrating the results of dCAPS analysis, immunoblot analysis and assays of starch synthase activity of granule-bound proteins indicates that the SNP in the Wx-D1 gene was responsible for its waxy character. This dCAPS marker is therefore useful as a marker to introduce the mutant allele into elite breeding lines.

Alleles↗

PubMed2006

Multiplexed SNP genotyping using nanobarcode particle technology.

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

DNA↗

PubMed2007

A functional SNP in ITIH3 is associated with susceptibility to myocardial infarction.

Myocardial infarction (MI) results from complex interactions of multiple genetic and environmental factors. To disclose genetic backgrounds of MI, we performed a large-scale, case-control association study using 52,608 gene-based single-nucleotide polymorphism (SNP) markers, and identified a candidate SNP located on chromosome 3p21.2-p21.1. Subsequent linkage-disequilibrium mapping indicated very significant association between MI and a SNP in exon 2 of the inter-alpha (globulin) inhibitor 3 gene (ITIH3; chi(2) = 24.88, P = 6.1 x 10(-7), 3,353 affected individuals versus 3,807 controls). In vitro functional analyses showed that this SNP enhanced the transcriptional level of the ITIH3 gene. Furthermore, we found expression of the ITIH3 protein in the vascular smooth muscle cells and macrophages in the human atherosclerotic lesions, suggesting ITIH3 SNP to be a novel genetic risk factor of MI.

Alpha-Globulins↗

PubMed2001

The power of genome-wide association studies of complex disease genes: statistical limitations of indirect approaches using SNP markers.

Genome-wide association studies using a dense map of single nucleotide polymorphism (SNP) markers seem to enable us to detect a number of complex disease genes. In such indirect association studies, whether susceptibility genes can be detected is dependent not only on the degree of linkage disequilibrium between the disease variant and the SNP marker but also on the difference in their allele frequencies. These factors, as well as penetrance of the disease variant, influence the statistical power of such approaches. However, the power of indirect association studies is not well understood. We calculated the number of individuals necessary for the detection of the disease variant in both direct and indirect association studies with a case-control design. The result shows that a remarkable reduction in the statistical power of indirect studies, compared with that of direct ones, is unavoidable in the genome-wide screening of complex disease genes. If there is a large difference in allele frequency between the disease variant and the marker, the disease variant cannot be detected. Because the frequency of the disease variant is unknown, SNP markers with various allele frequencies, or a large number of SNP markers, must be used in indirect association studies. However, if the number of SNP markers is increased, the obtained P value may not reach the significance level due to the Bonferroni adjustment. Thus, to test a possible association between functional variants and a complex disease directly, we should identify such SNPs in as many genes as possible for use in genome-wide association studies.

Gene Frequency↗

PubMed1993

Differential responses in cyclic GMP formation induced by excitatory amino acids (EAA) and sodium nitroprusside (SNP) in various regions of the brain and of rats of varied age.

1. Sodium nitroprusside (SNP, 100 microM) caused a rapid and great increase of formation of cGMP in rat cerebellar slices. This effect was not blocked by L-NMMA (a NO synthetase inhibitor) or antagonists of the NMDA receptor complex (e.g. AP5 or MK 801). 2. Similarly, NMDA (100 microM) and glutamate (1 mM) caused a rapid but less significant increase of cGMP formation. This increase was blocked by NMDA receptor complex blockers (e.g. AP5, MK801 and kynurenate), and L-NMMA and L-nitroarginine. 3. In rats aged 12 days, both NMDA and kainate (at 100 microM) caused significantly increased levels of cGMP in the cerebellum, pons and medulla areas, whereas no significant alterations were found in the cerebral cortex, hippocampus or midbrain areas. 4. NMDA (100 microM) and SNP (300 microM) induced greater increases of cGMP in cerebellar slices in young (aged 13 days) animals than older ones of either sex. This effect decreased greatly after 35 days of age. In adult (2 months) animals the effect of NMDA had virtually disappeared whereas SNP was barely significantly present. 5. Our results suggest that brain region and age, but not sex, affected formation of cGMP induced by excitatory amino acids (EAA) and SNP. Furthermore, endogenous NO production is required by EAA, but not by SNP, in the formation of cGMP.

Aging↗

PubMed2005

Allele frequencies for 70 autosomal SNP loci with U.S. Caucasian, African-American, and Hispanic samples.

189 samples from 3 different U.S. sample groups Caucasian (74), African American (71) and Hispanic (44) were typed for 70 autosomal genetic markers. These 70 markers are bi-allelic (C/T) short nucleotide polymorphisms (SNPs). For each sample, the 70 SNP markers were typed in 11 unique 6-plexes and a single 4-plex PCR. A total of 10 of the 210 tests (70 loci x 3 populations) for Hardy-Weinberg equilibrium indicated a statistically significant result. In order to evaluate the minimum number of SNP loci needed to distinguish all 189 samples from one another, we ranked the loci according to their levels of observed heterozygosity and p-values obtained upon testing for Hardy-Weinberg equilibrium. The top 12 loci according to these ranking criteria were tabulated along with the number of unique genotypes observed when combining subsequent SNP markers. The 12 selected SNPs possessed an observed heterozygosity of >0.45 in all three populations examined and thus would be expected to exhibit more differences between samples. All of the 189 samples in this study were individualized with a subset of 12 SNP loci. However, it is likely that the addition of more than 12 SNP loci will be required to resolve larger sets of unrelated individuals from one another. By way of comparison, in these same 189 individuals all but one pair is resolved from one another with three of the traditional short tandem repeat (STR) loci possessing the highest heterozygosity values (D2S1338, D18S51, and FGA) run with the Identifiler kit. The final pair of unrelated samples could be resolved with the combination of 4 STR loci: D2S1338, D18S51, FGA, and VWA.

DNA Fingerprinting↗

PubMed2005

Validation of a 21-locus autosomal SNP multiplex for forensic identification purposes.

A single nucleotide polymorphism (SNP) multiplex has been developed to analyse highly degraded and low copy number (LCN) DNA template, i.e. <100 pg, for scenarios including mass disaster identification. The multiplex consists of 20 autosomal non-coding loci plus Amelogenin for sex determination, amplified in a single tube PCR reaction and visualised on the Applied Biosystems 3100 capillary electrophoresis (CE) system. Allele-specific primers tailed with shared universal tag sequences were designed to speed multiplex design and balance the amplification efficiencies of all loci through the use of a single reverse and two differentially labelled allele denoting forward universal primers. As the multiplex is intended for use with samples too degraded for conventional profiling, a computer program was specifically developed to aid interpretation. Critical factors taken into account by the software include empirically determined extremes of heterozygous imbalance (Hb) and the drop-out threshold (Ht) defined as the maximum peak height of a surviving heterozygous allele, where its partner may have dropped out. The discrimination power of the system is estimated at 1 in 4.5 million, using a White Caucasian population database. Comparisons using artificially degraded samples profiled with both the SNP multiplex and AMPFISTR SGM plus (Applied Biosystems) demonstrated a greater likelihood of obtaining a profile using SNPs for certain sample types. Saliva stains degraded for 147 days generated an 81% complete SNP profile whilst short tandem repeats (STRs) were only 18% complete; similarly blood degraded for 243 days produced full SNP profiles but only 9% with STRs. Reproducibility studies showed concordance between SNP profiles for different sample types, such as blood, saliva, semen and hairs, for the same individual, both within and between different DNA extracts.

Amelogenin↗