Genotyping of single nucleotide polymorphisms using base-quenched probe: A method does not invariably depend on the deoxyguanosine nucleotide

Most available methods for detecting single nucleotide polymorphisms (SNPs) are based principally on the system that can produce an increased fluorescence signal during hybridization. In the current study, we demonstrate a method of base-quenched probe for polymerase chain reaction (PCR) genotyping that requires only a pair of primers and one fluorescent probe and does not invariably depend on the deoxyguanosine nucleotide. This method further exploits the phenomenon of fluorescence quenching of fluorescent-labeled probe during hybridization to its complementary target gene’s sequence. 6-Carboxyfluorescein (FAM) can be directly conjugated to a base of either adenine (A), thymine (T), cytosine (C), or guanine (G), referred to as A-, T-, C-, or G-quenched probe, respectively, at either the 5′ or 3′ end. For describing the method in detail, we chose apolipoprotein M (apoM) as a target gene in the current study. DNA sequencing analyses validated that all four types of base-quenched probes could provide unbiased genotyping results (K = 1, P = 0.000), although the maximum speed of fluorescence increase, max(dF/dT), when using the G-quenched probe method, was approximately twofold lower than the others (P < 0.0001). Moreover, we applied this method to detect another seven SNPs in the genomes of phospholipase A2, monocyte chemoattractant protein 1 (MCP1), and l-ficolin, further confirming our method. It is concluded that this method is precise, simple, and economic as well as suitable for large-scale genotyping studies.     

High fidelity of whole-genome amplified DNA on high-density single nucleotide polymorphism arrays

Current microarray technology allows researchers to genotype a large number of SNPs with relatively small amounts of DNA. Nevertheless, researchers and clinicians still frequently face the problem of acquiring enough high-quality DNA for analysis. Whole-genome amplification (WGA) methods offer a solution for this problem, and earlier studies have shown that WGA samples perform reasonably well in small-scale genetic analyses (e.g. Affymetrix 10K array). To determine the performance of WGA products on a large-scale genotyping array, we compared the Affymetrix 250K array genotyping results of genomic DNA and their WGA products from four individuals. Our results indicate that WGA product performs well on the 250K array compared to genomic DNA, especially when using the BRLMM calling algorithm. WGA samples have high call rates (97.5% on average, compared to 99.4% for genomic DNA) and excellent concordance rates with their corresponding genomic DNA samples (98.7% on average). In addition, no apparent systematic genomic amplification bias can be detected. This study demonstrates that, although there is a slight decrease in the total call rates, WGA methods provide a reliable approach for increasing the amount of DNA samples for use with a common SNP genotyping array.     

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.     

A dual-probe hybridization method for reducing variability in single nucleotide polymorphism analysis with oligonucleotide microarrays

DNA microarray technology has become powerful and popular in mutation/single nucleotide polymorphism (SNP) discovery and genotyping. However, this method is often associated with considerable signal noise of nonbiological origin that may compromise the data quality and interpretation. To achieve a high degree of reliability, accuracy, and sensitivity in data analysis, an effective normalization method to minimize the technical variability is highly desired. In the current study, a simple and robust normalization method is described. The method is based on introduction of a reference probe coimmobilized with SNP probes on the microarray for a dual-probe hybridization (DPH) reaction. The reference probe is used as an intraspot control for the customized microarrays. Using this method, the interassay coefficient of variation (CV) was reduced significantly by approximately 10%. After DPH normalization, the CVs and ranges of the ratios were reduced by two to five times. The relative magnitudes of variation of different sources were also analyzed by analysis of variance. Glass slides were shown to contribute the most to the variance, whereas sampling and residual errors had relatively modest contribution. The results showed that this DPH-based spot-dependent normalization method is an effective solution for reducing experimental variation associated with microarray genotyping data.     

Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction

Different methods have been developed for single nucleotide polymorphism (SNP) typing during recent years. Allele-specific polymerase chain reaction (ASPCR) is a cost-saving method that scores SNPs by difference of the PCR efficiency of allele-specific primers. However, ASPCR for SNP typing is notoriously confounded for its locus-specific unpredictability and the laborious gel electrophoresis. In the current study, we investigated the real-time kinetics of ASPCR and found that a simple touchdown thermocycling protocol improved its specificity significantly. Combined with real-time PCR, we developed a homogeneous genotyping method and scored more than 1000 genotypes, including all transition and transversion SNPs. A clear genotyping result was identified and validated the robustness of the method. Optimization of reactions and intrinsic modification of allele-specific primers, a laborious process but one that is repeatedly reported to be inevitable for successful ASPCR, was proved to be unnecessary with our method. Accuracy was confirmed with mass spectrometry. These characters enabled real-time ASPCR with the touchdown thermocycling protocol being very competitive among various SNP typing methods for large-scale genetic studies.     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

PCR amplification on magnetic nanoparticles: application for high-throughput single nucleotide polymorphism genotyping

A novel approach for the genotyping of single nucleotide polymorphisms (SNPs) based on solidphase PCR on magnetic nanoparticles (MNPs) is described. PCR products were amplified directly on MNPs. The genotypes of a given SNP were differentiated by hybridization with a pair of allele-specific probes labeled with dual-color fluorescence (Cy3, Cy5). The results were analyzed by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. Electrophoresis analysis indicated that PCR could proceed successfully when MNPs-bound primers were used. Furthermore, nine different samples were genotyped and their fluorescent signals were quantified. Genotyping results showed that three genotypes for the locus were very easily discriminated. The fluorescent ratios (match probe:mismatch probe signal) of homozygous samples were over 9.3, whereas heterozygous samples had ratios near 1.0. Without any purification and concentration of PCR products, this new MNP-PCR based genotyping assay potentially provides a rapid, labor-saving method for genotyping of a large number of individuals.     

DNA-probes for the highly sensitive identification of single nucleotide polymorphism using single-molecule spectroscopy

This article presents a new, highly sensitive method for the identification of single nucleotide polymorphisms (SNPs) in homogeneous solutions using fluorescently labeled hairpin-structured oligonucleotides (smart probes) and fluorescence single-molecule spectroscopy. While the hairpin probe is closed, fluorescence intensity is quenched due to close contact between the chromophore and several guanosine residues. Upon hybridization to the respective target SNP sequence, contact is lost and the fluorescence intensity increases significantly. High specificity is achieved by blocking sequences containing mismatch with unlabeled oligonucleotides. Time-resolved single-molecule fluorescence spectroscopy enables the detection of individual smart probes passing a small detection volume. This method leads to a subnanomolar sensitivity for this single nucleotide specific DNA assay technique.     

MagSNiPer: a new single nucleotide polymorphism typing method based on single base extension, magnetic separation, and chemiluminescence

We have developed a new method for typing single nucleotide polymorphisms (SNPs), MagSNiPer, based on single base extension, magnetic separation, and chemiluminescence. Single base nucleotide extension reaction is performed with a biotinylated primer whose 3' terminus is contiguous to the SNP site with a tag-labeled ddNTP. Then the primers are captured by magnetic-coated beads with streptavidin, and unincorporated labeled ddNTP is removed by magnetic separation. The magnetic beads are incubated with anti-tag antibody conjugated with alkaline phosphatase. After the removal of excess conjugates by magnetic separation, SNP typing is performed by measuring chemiluminescence. The incorporation of labeled ddNTP is monitored by chemiluminescence induced by alkaline phosphatase. MagSNiPer is a simple and robust SNP typing method with a wide dynamic range and high sensitivity. Using MagSNiPer, we could perform SNP typing with as little as 10(-17) mol of template DNA.     

Association of cytotoxic T lymphocyte-associated antigen 4 gene single nucleotide polymorphism with type 1 diabetes mellitus in Madurai population of Southern India

Type 1 diabetes mellitus formerly called juvenile diabetes, is an organ specific T-cell mediated autoimmune disease characterized by the progressive loss of function of the insulin producing beta-cells of the islets of Langerhans. Cytotoxic T lymphocyte-associated antigen 4 gene (CTLA-4) has been proposed as a candidate gene for conferring susceptibility to autoimmunity. Association of CTLA-4 gene polymorphism is well established in autoimmune endocrinopathies across world population. The present study was conducted to investigate the association of CTLA-4 exon 1 49A/G polymorphism with TIDM in Madurai, a city in Southern India. Fifty three clinically proven T1DM patients and 53 control subjects with no history of autoimmune disease were recruited for the study. Genomic DNA was extracted from peripheral blood. CTLA-4 exon 1 49 A/G polymorphism was assessed using PCR-RFLP methods. Our findings revealed a significant association of CTLA-4 exon 1 49 A/G polymorphism with T1DM in Madurai population.     

Association study of CCR6 rs3093024 with Rheumatoid Arthritis in a Pakistani cohort

C-C Chemokine receptor 6 (CCR6), an important protein in inflammatory and immunological responses, has been previously reported to be associated with rheumatoid arthritis (RA). Therefore, in order to replicate these findings, a case-control study was conducted on 500 subjects (including 250 RA patients and 250 healthy controls) of Pakistani origin. The aim of this study was to determine the association of CCR6 rs3093024 variant with RA and identify its role in splicing events using bioinformatics tools. The clinical and demographic characteristics of the patients were collected using a well-designed questionnaire. The genotype frequencies of CCR6 rs3093024 variant were determined using tetra-primer ARMS-PCR (amplification of refractory mutation system-polymerase chain reaction) method. A significant difference was found between CCR6 rs3093024 genotype frequencies [P = 0.0016, χ² = 12.915]. Similarly, a significant difference in the allele frequencies between RA patients and healthy controls was also observed [P = 0.0003 and OR (95% CI) = 0.63 (0.49–0.80)]. The stratification of patients showed that there was a significant increase in AA genotype against AG + GG in patients [P = 0.0014, OR (95% CI) = 2.0 (1.32–3.02)]. Furthermore, using bioinformatics analysis, it was found that CCR6 rs3093024 variant might create a potential splicing enhancer motif (SF2/ASF (IgM-BRCA1) with score of 77.92; Threshold 70.53), which might have important impact on the product of this gene. This study suggests that the A variant of CCR6 rs3093024 variant is significantly associated with RA-risk and its G variant is protective in Pakistani population but a multi-cohort large sized population study is needed to elucidate these results. Moreover, functional studies are needed to highlight the effects of this polymorphism on the function of CCR6 gene.     

The pattern of clinical breast cancer metastasis correlates with a single nucleotide polymorphism in the C1qA component of complement

Complement is one of primary defense mechanisms against intravascular microorganisms and could play a role in the immune response to malignancy and hence its clinical behavior. We evaluated if the sole coding polymorphism of C1qA associates with outcome in patients with breast carcinoma. Genotyping for C1qA_[276A/G] was performed in 63 breast cancer subjects with localized tumor and compared with that in 38 breast cancer subjects with metastasis. Established risk factors for clinical outcome were considered and evaluated in multivariable analysis. Breast cancer subjects with heterozygous or homozygous C1qA_[276G] genotype had a higher rate of metastasis than subjects with the homozygous C1qA_[276A] genotype [hazard ratio (HR) 2.4, 95% confidence interval (CI) 1.1–4.1]. This association was stronger when only metastatic sites associated with hematogenous spread, i.e., to the bone, liver, and brain, were considered (HR 3.5, 95% CI 1.4–5.6) and remained statistically significant after adjustment for the number of positive lymph nodes, estrogen receptor status, and progesterone receptor status. There was no statistical difference in the C1qA_[276A/G] allelic distribution between all subjects with breast cancer and controls. These results suggest there could be an association of a single nucleotide polymorphism at position 276 of the C1qA component of complement with breast cancer metastasis to sites linked to hematogenous spread of disease. The C1qA polymorphism associated with decreased distant metastasis has also been correlated with an increased incidence of subcutaneous systemic lupus and C1q deficiencies, suggesting that an altered immune response may play a role in the observed association. Supported in part by National Institute of Health grant R21-CA90822, Friends You Can Count On! grant 1-87093-00, and the Woody and Louise White Cancer Research Fund.     

Six diagnostic single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trouts

Ten primer pairs were screened to develop single nucleotide polymorphism (SNP) TaqMan assays that will distinguish California golden trout and some rainbow trouts (Oncorhynchus mykiss sspp., O. m. aguabonita) from the Paiute and Lahontan cutthroat trouts (Oncorhynchus clarkii seleniris, O. c. henshawi). From these 10 primer pairs, one mitochondrial and five nuclear fixed SNP differences were discovered and developed into TaqMan assays. These six assays will be useful for characterizing and monitoring hybridization between these groups. Additional Oncorhynchus clarkii sspp. and Oncorhynchus mykiss sspp. were assayed to determine if these assays are useful in closely related species.     

Simultaneous genotyping of multiplex single nucleotide polymorphisms of the K-ras gene with a home-made kit

Accurate and fast genotyping of single nucleotide polymorphisms (SNPs) is important in the human genome project. Here an automated fluorescent method that can rapidly and accurately genotype multiplex known SNPs was developed by using a homemade kit, which has lower cost but higher resolution than commercial kit. With this method, oncogene K-ras was investigated, four known SNPs of K-ras gene exon 1 in 31 coloerctal cancer patients were detected. Results indicate that mutations were present in 8(26%) of 31 patients, and most mutations were localized in codon 12. The presence of these mutations is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Relationship between single nucleotide polymorphism of interleukin-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population

Interleukin-18 (IL-18) is a multi-functional cytokine capable of inducing either Th1 or Th2 polarization depending on the immunologic milieu. IL-18 may influence the host response to Mycobacterium tuberculosis (M.tb) infection. To investigate the relationship between single nucleotide polymorphisms of the IL-18 and susceptibility to pulmonary tuberculosis in the Chinese Han population, the IL-18 gene was sequenced to detect polymorphisms and to examine the genotype frequencies in 300 patients and 702 healthy controls. DNA sequencing revealed three IL-18 variants: rs1946518, rs5744247, and rs549908. It also revealed that allele A of rs1946518 confers a 1.47-fold increased risk of developing tuberculosis (TB) (P = 0.0001, OR [95%CI] = 1.47 [1.21-1.78]), and that the C allele of rs5744247 confers a 0.77-fold decreased risk of disease (P = 0.01, R [95%CI] = 0.77 [0.632-0.937]). The genotypes rs1946518, rs5744247 and rs549908 were found to be significantly associated with TB. Estimation of the frequencies of haplotypes revealed a potential risk haplotype AGA (P = 0.01, OR [95%CI] = 1.41 [1.15-1.72]) and a protective haplotype CCA (P = 0.01, OR [95%CI] = 0.70 [0.57-0.85]) for TB. The present findings suggest that polymorphisms in the IL-18 gene may affect susceptibility to TB and increase the risk of developing the disease in the Chinese Han population.     

Association of cytokine gene polymorphisms with malignant melanoma in Caucasian population

It has been hypothesized that polymorphisms expected to result in functional changes in cytokine genes may influence susceptibility to cancer, including malignant melanoma (MM). Here, we have screened 24 potentially functional polymorphisms in five cytokine genes by PCR-SBT and PCR-SSP methods in 122 MM cell lines derived from Caucasian patients. The polymorphic positions studied were: TNFA −1031, −863, −857, −851, −574, −376, −308, −238, +488; TGFB1 −988, −800, −509, +869, +915, +652, +673, +713, +788; IL10 −1082, −819, −592; IL6 −174; IFNG −333, +874. The frequencies of cytokine genotypes of melanoma tumours were compared with those published for healthy Caucasians. It was found that TNFA −238 GA, TGFB1 −509 CT, −800 GG, IFNG +874 AT, IL6 −174 GG, IL10 −1082 GA genotypes were significantly decreased, while TNFA −238 AA, −857 CC, TGFB1 −509 TT, IFNG +874 AA, IL6 −174 CC, IL10 −1082 AA, −819 TT, −592 AA genotypes were significantly increased, in MM. This suggests that genotypes provisionally associated with low expression of pro-inflammatory and immunomodulatory TNF-α, IFN-γ and IL-6 and anti-inflammatory IL-10 and TGF-β1 could be involved in the mechanisms of cancer progression and escape from immunoserveilance.     

Association between single nucleotide polymorphisms of TPH1 and TPH2 genes,and depressive disorders

Tryptophan catabolites pathway disorders are observed in patients with depression. Moreover, single nucleotide polymorphisms of tryptophan hydroxylase genes may modulate the risk of depression occurrence. The objective of our study was to confirm the association between the presence of polymorphic variants of TPH1 and TPH2 genes, and the development of depressive disorders. Six polymorphisms were selected: c.804‐7C>A (rs10488682), c.‐1668T>A (rs623580), c.803+221C>A (rs1800532), c.‐173A>T (rs1799913)—TPH1, c.‐1449C>A (rs7963803), and c.‐844G>T (rs4570625)—TPH2. A total of 510 DNA samples (230 controls and 280 patients) were genotyped using TaqMan probes. Among the studied polymoorphisms, the G/G genotype and G allele of c.804‐7C>A—TPH1, the T/T homozygote of c.803+221C>A—TPH1, the A/A genotype and A allele of c.1668T>A—TPH1, the G/G homozygote and G allele of c.‐844G>T—TPH2, and the C/A heterozygote and A allele of c.‐1449C>A—TPH2 were associated with the occurrence of depression. However, the T/T homozygote of c.‐1668T>A—TPH1, the G/T heterozygote and T allele of c.‐844G>T—TPH2, and the C/C homozygote and C allele of c.‐1449C>A—TPH2 decreased the risk of development of depressive disorders . Each of the studied polymorphisms modulated the risk of depression for selected genotypes and alleles. These results support the hypothesis regarding the involvement of the pathway in the pathogenesis of depression.     

Association of a single nucleotide polymorphism in the akirin 2 gene with economically important traits in Korean native cattle

The akirin 2 gene, located on chromosome 9 in cattle, was previously reported to be associated with nuclear factor‐kappa B (NF‐κB), involved in immune reactions and marbling of meat. To determine whether a single nucleotide polymorphism (SNP) in akirin 2 is associated with economically important traits of Korean native cattle, the c.*188G>A SNP DNA marker in the 3′‐UTR region of akirin 2 was analyzed for its association with carcass weight, longissimus muscle area and marbling. The c.*188G>A SNP was genotyped by polymerase chain reaction restriction fragment length polymorphism, and the frequency of the AA, AG, and GG genotypes were 6.82%, 71.29% and 21.88% respectively. This SNP was significantly associated with longissimus muscle area (Bonferroni corrected P < 0.05), and marbling score (Bonferroni corrected P < 0.01). These results suggest that the c.*188G>A SNP of akirin 2 might be useful as a DNA marker for longissimus muscle area and marbling scores in Korean native cattle.     

Association between polymorphisms in OAS2 and CD209 genes and predisposition to chronic hepatitis C in Russian population

Chronic hepatitis C is a severe liver disease caused by positive-strand RNA virus. Previously, we reported an association between seven single nucleotide polymorphisms (SNPs) in four innate immunity genes (OAS2, OAS3, CD209, and TLR3) and human predisposition to tick-borne encephalitis, caused by a virus from the same Flaviviridae family, in a Russian population. Currently, genotype and allele frequencies for these SNPs were analyzed in 75 chronic hepatitis C patients and compared with the population control (269 Novosibirsk citizens). Data obtained suggest that the OAS2 rs1293762 and CD209 rs2287886 SNPs are associated with predisposition to chronic hepatitis C in Russian population.     

Analysis of single nucleotide polymorphisms in the FAS and CTLA-4 genes of peripheral T-cell lymphomas

Angioimmunoblastic T-cell lymphoma (AILT) represents a subset of T-cell lymphomas but resembles an autoimmune disease in many of its clinical aspects. Despite the phenotype of effector T-cells and high expression of FAS and CTLA-4 receptor molecules, tumor cells fail to undergo apoptosis. We investigated single nucleotide polymorphisms (SNPs) of the FAS and CTLA-4 genes in 94 peripheral T-cell lymphomas. Although allelic frequencies of some FAS SNPs were enriched in AILT cases, none of these occurred at a different frequency compared to healthy individuals. Therefore, SNPs in these genes are not associated with the apoptotic defect and autoimmune phenomena in AILT.