Genotyping the -521C/T functional polymorphism in the promoter region of dopamine D4 receptor (DRD4) gene

The -521C/Tsingle nucleotide polymorphism (SNP) in the promoter region of the dopamine D4 receptor gene (DRD4) has recently been detected in oriental (Japanese) individuals and related to novelty seeking and schizophrenia. Here, we report the analysis of the -521C/T polymorphism in a Caucasian (Hungarian) population using two independent genotyping methods. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) procedure utilized the Fspl restriction site around the -521 position. An additional, nonpolymorphic cleavage site was also included into the amplified region to serve as an internal standard for verifying the completion of the digestion. As another independent method, a tetraprimer system for single-tube allele-specific PCR (SAS-PCR) was developed to generate -521C and -521T specific PCR products with different fragment sizes. Consequently, genotyping with SAS-PCR is based on the gel-electrophoretic separation of the allele-specific double-stranded DNA (dsDNA) fragments. 119 healthy Hungarian individuals were genotyped for -521C/T polymorphism of the dopamine D4 promoter region, using both methods. Similar allele frequencies were found (-521C allele: 0.43; -521T allele: 0.57) as reported earlier for the Japanese population.     

Genotyping and haplotyping of the dopamine D4 receptor gene by capillary electrophoresis

In this paper we report on simultaneous genotyping of adjacent polymorphisms (referred to as haplotyping) by combining double-tube allele-specific polymerase chain reaction, restriction fragment length polymorphism and capillary gel electrophoresis analysis of the resulting fragments. Direct molecular haplotyping is of particular importance in the case of double heterozygote samples, since in these instances the haplotype structure cannot be constructed based on genotype data. Our approach provided a powerful tool for coincidental genotype analysis of the 48 base pair (bp) variable number of tandem repeats of the third exon and haplotype investigation of the -616CG and -521CT single nucleotide polymorphisms of the dopamine D4 receptor (DRD4) gene. The linear polyacrylamide sieving matrix was optimized for the size range of the double-stranded DNA fragments of interest varying from 35 to 763 bp. We demonstrated that capillary gel electrophoresis in combination with laser induced fluorescence detection offers a sensitive and accurate tool for automated haplotyping in clinical settings.     

Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was screened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified genomic DNA fragments using intron-based primers. DNA samples with abnormal TGGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%): 496delGT (1), 499delTGTT (4), T528A = D176E (2), T539A = L180X (1), 540insA (1), C574T = R192X (1). Thus, a total of six different mutations was identified in exon 4b but none in exons 4a and 4c. Only the missense mutation D176E, which we assume to be a nonpathogenic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been described before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 (TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp deletion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that microlesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.     

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.     

Developing and population analysis of a new multiplex panel of 18 microhaplotypes and compound markers using next generation sequencing and its application in the Shaanxi Han population

Compound marker consists of two different types of genetic markers, like deletion/insertion polymorphism and single nucleotide polymorphism in the genomic region of 200 bp, and microhaplotype consists of a series of closely linked single nucleotide polymorphisms in a small DNA segment (<300 bp), which show great potential for human identifications and mixture analyses. In this study, we initially selected 23 novel genetic markers comprising 10 microhaplotypes and 13 compound markers according to previously reported single nucleotide polymorphism or deletion/insertion polymorphism loci. Genetic distributions of these 23 loci in different continental populations showed that they could be used as valuable loci for forensic human identification purpose. Besides, high informativeness values (>0.1) were observed in six loci which could be further employed for forensic ancestry analyses. Finally, 18 loci were successfully developed into a multiplex panel and detected by the next generation sequencing (NGS) technology. Further analyses of these 18 loci in the studied Shaanxi Han population showed that 15 loci exhibited relatively high expected heterozygosities (>0.5). Cumulative power of discrimination (0.999 999 999 99 4835) of these 18 loci revealed that the multiplex panel could also be utilized for human identifications in the studied Shaanxi Han population.     

Association of transforming growth factor-beta1 gene polymorphisms with a hepatocellular carcinoma risk in patients with chronic hepatitis B virus infection

Transforming growth factor-beta1 (TGF-beta1) can act as both a tumor suppressor and a stimulator of tumor progression. We have examined the relationship between polymorphisms of the TGF-beta1 gene and the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B virus (HBV) infection. A total of 1,237 Korean subjects were prospectively enrolled; 1,046 patients with chronic HBV infection and 191 healthy controls with no evidence of recent or remote HBV infection. The patients were divided into two groups: those without (n = 809) and those with HCC (n = 237). Single nucleotide polymorphisms (SNPs) of TGF-beta1 were searched for and genotyped using the single base extension method. In Korean subjects, only two SNPs were found among the seven known polymorphisms of TGF-beta1, at position -509 and in codon 10. The risk of HCC was significantly lower in patients with the T/T or C/T genotypes than in those with the C/C genotypes at position -509 (P < 0.02), and also lower among those with the Pro/Pro or Leu/Pro genotypes than in those with the Leu/Leu genotypes in codon 10 (P < 0.007). Haplotype analysis revealed that the possession of [-509C > T; L10P] conferred a decreased likelihood of HCC (OR = 0.74; 95% CI, 0.59-0.93; P = 0.008). In conclusion, the presence of the TGF-beta1 -509C > T promoter or of the L10P polymorphism, and the combination of both [-509C > T; L10P] as a haplotype were strongly associated with a reduced risk of HCC in patients with chronic HBV infection.     

Genotyping single nucleotide polymorphisms using intact polymerase chain reaction products by electrospray quadrupole mass spectrometry

Both single nucleotide polymorphisms (SNPs) and mutations are commonly observed in the gene encoding the tumor suppressor protein, p53. SNPs occur at specific locations within genes whereas mutations may be distributed across large regions of genes. When determining nucleotide differences, mass spectrometry is the only method other than Sanger sequencing which offers direct structural information. Electrospray ionization (ESI) quadrupole mass spectrometry (MS) analysis of intact polymerase chain reaction (PCR) products was performed following a simple purification and on-line heating to limit ion adduction. The PCR products were amplified directly from genomic DNA rather than plasmids, as in our previous work. Two known polymorphisms of the p53 gene were genotyped. A cytosine (C) or guanine (G) transversion, designated C <--> G (G <--> C on the opposite strand), were each detected by a 40.0 Da change upon ESI quadrupole MS analysis. Using known PCR products as standards, the genotypes determined for 10 human samples corresponded with restriction fragment length polymorphism (RFLP) analysis. Cytosine/thymine (T) transitions, designated C <--> T (G <--> A on the opposite strand), were also genotyped by ESI-MS. This SNP is discriminated by a 15.0 Da change on one strand (C <--> T) and a 16.0 Da change on the other (G <--> A). Appropriate sample preparation and instrumental configuration (including heated sample inlet syringe and MS source), to limit adducts, are both vital for successful ESI quadrupole MS analysis of intact PCR products.     

Improved Allele-specific Polymerase Chain Reaction for Single Nucleotide Polymorphism Genotyping

An improved allele-specific PCR(AS-PCR) approach was applied to investigating-55C/T polymorphism in promoter region of the uncoupling protein 3(UCP3)gene.AS-PCR is a competitive PCR method which is based on positioning the 3' base of a PCR primer to match one single nucleotide polymorphism(SNP) allele and accurately extend only the correctly matched primer.But it is limited in use because of its poor specificity.In this study,we improved the specificity of AS-PCR by introducing additional mismatch at the pe...     

Single nucleotide polymorphism detection in the hMSH2 gene using conformation-sensitive CE

CE allows for highly reproducible analysis of DNA fragments which can be used to detect DNA mutations including SNPs. We have utilized a simple and direct CE analysis method for SNP analysis called conformation-sensitive CE (CSCE), based on the principle of single nucleotide different to produce conformational changes in the mildly denaturing solvent system. This method was applied to analysis of a mutation in the promoter region of the hMSH2 gene. This gene belongs to the human DNA mismatch repair system, which is responsible for recognizing and repairing mispaired nucleotides, and mutations in the hMSH2 gene are known to cause hereditary nonpolyposis colorectal cancer (HNPCC). PCR fragments generated from the promoter region of the hMSH2 gene, displaying either a C/C homozygote, C/T heterozygote, or T/T homozygote genotype, did not require further pretreatment before electrokinetic injection. The CE separation, using a 1xTris-borate-EDTA (TBE) buffer containing 3% w/v hydroxylethyl cellulose (HEC) and 6 M urea, was performed under reverse polarity with a separation temperature of 15 degrees C. The genotypes of 204 healthy volunteers and 13 colorectal cancer patients were determined using CSCE, and the results confirmed by DNA sequencing. While the CSCE separations were shown to be highly reproducible and sensitive for screening large populations, no correlation was observed between cancer patients and this hMSH2 gene polymorphism.     

Sequence selective recognition in the minor groove of dsDNA by pyrrole,imidazole-substituted bis-benzimidazole conjugates

A series of pyrrole, imidazole-substituted bis-benzimidazole conjugates, Py-Py-Im-gamma-biBenz, Py-Py-gamma-biBenz, Py-Im-gamma-biBenz, and Im-Py-gamma-biBenz (1-4), were prepared in an attempt to target dsDNA sequences possessing both A/T and G/C bps. The dsDNA interactions and sequence specificity of the conjugates have been characterized via spectrofluorometric titrations and thermal melting studies. All conjugates form 1:1 complexes with dsDNA at subnanomolar concentrations. The Im moiety selectively recognizes a G/C bp embedded in the A/T-rich binding site. This represents the first clear example of sequence selective recognition in a 1:1 motif.(1) The equilibrium association constant (K(1)) for complexation of a specific nine-bp dsDNA site, 5'-gcggTATGAAATTcgacg-3', by conjugate 1 is approximately 2.6 x 10(9) M(-1). Displacement of the G/C position or G/C-->A/T substitution within the nine-bp site decreases the K(1) by approximately 8-fold, whereas two continuous G/C bps decrease the K(1) by approximately 50-fold magnitude. The K(1) values for seven-bp dsDNA, 5'-gcggtaTGAAATTcgacg-3' and 5'-gcggtaCAAAATTcgacg-3', binding sites by conjugates Py-Im-gamma-biBenz (3) and Im-Py-gamma-biBenz (4) are approximately 2.3 x 10(9) and approximately 1.2 x 10(9) M(-1), respectively. However, the conjugates with no Im moiety, Py-Py-gamma-biBenz (2) and Py-Py-Py-gamma-biBenz (5 and 6), are specific for seven- to nine-bp A/T-rich sites and single A/T-->G/C bp substitution within the binding site decreases the K(1) values by 1-2 orders of magnitude.     

A single-strand conformation polymorphism method for the large-scale analysis of mutations/polymorphisms using capillary array electrophoresis

We present a high-throughput single-strand conformation polymorphism (SSCP) method, performed on a commercially available capillary array DNA sequencer. We tested various sieving matrices and electrophoretic conditions, using 51 DNA fragments which included 45 fragments carrying only one single nucleotide polymorphism (SNP), 4 fragments having two SNPs and 2 fragments with insertion or deletion. Resolution of alleles was improved by increasing concentrations of both sieving matrices and buffers, and all examined polymorphisms of DNA fragments were detected, most of them (45 fragments) as clearly split allele peaks in heterozygotes. Allele frequencies of SNPs can be estimated accurately by determining the relative amounts of alleles in pooled DNA. In this method, the turn-around time for the analysis of 96 samples is less than 3 h. These results demonstrate that capillary array-based SSCP is an efficient and accurate technique for the large-scale quantitative analysis of mutations/polymorphisms.     

A lateral flow biosensor for detection of single nucleotide polymorphism by circular strand displacement reaction

A lateral flow biosensor for detection of single nucleotide polymorphism based on circular strand displacement reaction (CSDPR) has been developed. Taking advantage of high fidelity of T4 DNA ligase, signal amplification by CSDPR, and the optical properties of gold nanoparticles, this assay has reached a detection limit of 0.01 fM.     

Analysis of Single Nucleotide Polymorphism in Human Paraoxonase 1 Gene（Q192R） with Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry

Introduction Singlenucleotidepolymorphisms(SNPs)arethe mostabundantDNAmarkersinthehumangenomeoc curringatafrequencyofoneinevery500—1000nu cleotides[1].Avarietyofmethodshavebeenusedfor theanalysisofsinglenucleotidepolymorphisms,inclu dingrestrictionfragme…     

Electron transfer in DNA from guanine and 8-oxoguanine to a radical cation of the carbohydrate backbone

Photolysis of a 4'-pivaloyl-substituted nucleotide in single- and double-stranded DNA (1) generated an enol ether radical cation 4 that was reduced to enol ether 17 by electron transfer from the nearest guanoside (G). Variation of the nucleotide sequence demonstrated a strong distance dependence of this electron-transfer rate with beta = 1.0 +/- 0.1 A(-1). When 8-oxoguanosine (G(oxo)) was used as the electron donor, the rate of the electron transfer increased by a factor of 4 but the distance dependence of the transfer remained unchanged within experimental error. In single strands, the number of intervening A, T, and C nucleotides had a much smaller effect; the rate remained nearly constant for two, three, or four intervening nucleotides. This is explained by the flexibility of the single-stranded oligonucleotides.     

Geometries and electronic properties of carbon-nitrogen clusters C8N4 and C8N4+/-

The geometry optimization and frequency analysis for the low-lying electronic states of C(8)N(4) and its ions are performed at the DFT/6-31G(d) level. Their energies are calibrated at the CCSD(T)/6-31G(d) level of theory. Ionization energy, electron affinity, binding energy of C(8)N(4), and anion photoelectron spectra of C(8)N(4)(-) are provided at the CCSD(T)/6-31G(d) level. Mulliken populations, leading configurations, bond orders, and compositions of molecular orbitals are used to examine the bonding characteristics in the low-lying electronic states of C(8)N(4) and its ions. It is surprising to find that the ground state of C(8)N(4) is the open shell (5)A(1) state. Interestingly for the low-lying electronic states of C(8)N(4) and its ions, their structures significantly corrugated, which may be caused by their larger [N]/([N]+[C]) ratios. In addition, the similarities and differences between C(8)N(4) and C(10)N(2)(II) are analyzed and discussed.     

Quartz crystal microbalance detection of DNA single-base mutation based on monobase-coded cadmium tellurium nanoprobe

A new method for the detection of point mutation in DNA based on the monobase-coded cadmium tellurium nanoprobes and the quartz crystal microbalance (QCM) technique was reported. A point mutation (single-base, adenine, thymine, cytosine, and guanine, namely, A, T, C and G, mutation in DNA strand, respectively) DNA QCM sensor was fabricated by immobilizing single-base mutation DNA modified magnetic beads onto the electrode surface with an external magnetic field near the electrode. The DNA-modified magnetic beads were obtained from the biotin-avidin affinity reaction of biotinylated DNA and streptavidin-functionalized core/shell Fe(3)O(4)/Au magnetic nanoparticles, followed by a DNA hybridization reaction. Single-base coded CdTe nanoprobes (A-CdTe, T-CdTe, C-CdTe and G-CdTe, respectively) were used as the detection probes. The mutation site in DNA was distinguished by detecting the decreases of the resonance frequency of the piezoelectric quartz crystal when the coded nanoprobe was added to the test system. This proposed detection strategy for point mutation in DNA is proved to be sensitive, simple, repeatable and low-cost, consequently, it has a great potential for single nucleotide polymorphism (SNP) detection.     

High-performance liquid chromatography determination of nitrated polycyclic aromatic hydrocarbons by indirect fluorescence detection

A high-performance liquid chromatography (HPLC) method for the analysis of nitrated polcyclic aromatic hydrocarbons (NPAHs) is reported. NPAH mixtures were pre-concentrated using solid-phase extraction and well resolved on a C(18) column. They were detected using an indirect method involving the quenching of the emission from the fluorophores 5,6,7,8-tetrahydronaphthol (5,6,7,8-THN-1-OH), 7-amino-4-methyl coumarin (Coumarin 120, COU-120) and 3-hydroxy-4-(2-hydroxy-4-sulfo-1-naphthylazo)2-naphthalene carboxylic acid (Calcon carboxylic acid, CCA). Linear calibration curves were obtained in the range 1.1 x 10(-9) to 1.1 x 10(-8) mol/L. Using COU 120 as the fluorophore, the detection limit was 2.9 x 10(-10) mol/L for 1-nitronaphthalene and 2.1 x 10(-11) mol/L for 2-nitrofluorene. Recoveries of NPAHs from spiked tap water samples were between 88 and 100%.     

Accurate ab initio structure, dissociation energy, and vibrational spectroscopy of the F(-)-CH4 anion complex

Accurate equilibrium structure, dissociation energy, global potential energy surface (PES), dipole moment surface (DMS), and the infrared vibrational spectrum in the 0-3000 cm(-1) range of the F(-)-CH4 anion complex have been obtained. The equilibrium electronic structure calculations employed second-order M?ller-Plesset perturbation theory (MP2) and coupled-cluster (CC) method up to single, double, triple, and perturbative quadruple excitations using the aug-cc-p(C)VXZ [X = 2(D), 3(T), 4(Q), and 5] correlation-consistent basis sets. The best equilibrium geometry has been obtained at the all-electron CCSD(T)/aug-cc-pCVQZ level of theory. The dissociation energy has been determined based on basis set extrapolation techniques within the focal-point analysis (FPA) approach considering (a) electron correlation beyond the all-electron CCSD(T) level, (b) relativistic effects, (c) diagonal Born-Oppenheimer corrections (DBOC), and (d) variationally computed zero-point vibrational energies. The final D(e) and D0 values are 2398 +/- 12 and 2280 +/- 20 cm(-1), respectively. The global PES and DMS have been computed at the frozen-core CCSD(T)/aug-cc-pVTZ and MP2/aug-cc-pVTZ levels of theory, respectively. Variational vibrational calculations have been performed for CH4 and F(-)-CH4 employing the vibrational configuration interaction (VCI) method as implemented in Multimode.     

交河故城古车师人的线粒体DNA分析

从距今2000～2500年左右的交河故城古代人骨中提取古DNA,用4对重叠引物对线粒体基因组的调控区(363bp)进行了扩增及测序.线粒体基因组编码区的扩增片段用于限制性片段多样性分析.结果显示4个个体中具有3个DNA序列,其中来自不同墓穴的两个个体的序列相同,说明这两者间有密切的母系遗传关系.系统发育分析表明古车师的这4个个体分散分布在现代新疆维吾尔人的序列之中.从这些结果可以初步得出结论,即古车师人群并不是一个同源群体,在早期铁器时代,欧亚人群的混合就已经存在了.