Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization of 12 single nucleotide polymorphisms (SNPs) in Pacific abalone, Haliotis discus hannai

We report here for the first time 12 polymorphic single nucleotide polymorphisms (SNPs) in a commercially important gastropod, Pacific abalone (Haliotis discus hannai) that were identified by searching expressed sequence tag database. These SNP loci (seven nuclear and five mitochondrial SNPs) were polymorphic among 37 wild abalone individuals, based on a four-primer allele-specific polymerase chain reaction analysis. All loci had two alleles and the minor allele frequency ranged from 0.027 to 0.473. For the seven nuclear SNPs, the expected and observed heterozygosities ranged from 0.053 to 0.499 and from 0.054 to 0.811, respectively.

Mining expressed sequences for single nucleotide polymorphisms in Pacific abalone Haliotis discus hannai

Although single nucleotide polymorphisms (SNPs) are important resources for population genetics, pedigree analysis and genomic mapping, such loci have not been reported in Pacific abalone so far. In this study, a bioinformatics strategy was adopted to discover SNPs within the expressed sequences (ESTs) of Pacific abalone, Haliotis discus hannai, and furthermore, polymerase chain reaction direct sequencing (PCR-DS) and allele-specific PCR (AS-PCR) were used for SNPs detection and genotype scoring respectively. A total of 5893 ESTs were assembled and 302 putative SNPs were identified. The average density of SNPs in ESTs was 1%. Fifty-two sets of sequencing primers were designed from SNPs flanking ESTs to amplify the genomic DNA, and 13 could generate products of expected size. Polymerase chain reaction direct sequencing of the amplification products from pooled DNA samples revealed 40 polymorphic SNP loci. Using a modified tetra-primer AS-PCR, seven mitochondrial and six nuclear SNPs were typed and characterized among 37 wild abalones. In conclusion, it is feasible to discover SNPs from number limited ESTs and the AS-PCR as a simple, robust and reliable assay could be a primary method for small- and medium-scale SNPs detection in abalones as well as other non-model organisms.

Detection of transgenic DNA in tilapias (Oreochromis niloticus, GIFT strain) fed genetically modified soybeans (Roundup Ready)

We used nested-polymerase chain reaction (PCR) to detect Roundup Ready soybean in aquatic feeds and feeding tilapias. A template concentration of 10(-10) g mu L-1 DNA solution could be detected with a dilute degree of 0.01%. Most (90.6%) of the aquatic feeds containing soybean byproduct included exogenous DNA segments. We also compared genetically modified (GM) soybean with non-GM soybean diets in feeding tilapias (Oreochromis niloticus, GIFT strain) and examined the residual fragments (254 bp) of GM soybeans. Tilapias receiving GM soybean diets had DNA fragments in different tissues and organs, indicating that exogenous GM genes were absorbed systemically and not completely degraded by the tilapia's alimentary canal.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Chromosomal mapping of the major ribosomal RNA genes in the dwarf surfclam (Mulinia lateralis Say)

Chromosomal location of the major ribosomal RNA genes (rRNA) were studied in the dwarf surfclam (Mulinia lateralis, Say) using fluorescence in situ hybridization (FISH). FISH probes for the rRNA genes were made by polymerase chain reaction (PCR), labeled with digoxigenin-11-dUTP and detected with fluorescein-labeled antidigoxigenin antibodies. Mulinia lateralis had a diploid number of 38 chromosomes and all chromosomes were telocentric. FISH with the rRNA probe produced positive and consistent signals on two pairs of chromosomes: Chromosome 15 with a relative length of 4.6% and Chromosome 19, the shortest chromosome. Both loci were telomeric. The rRNA location provides the first physical landmark of the M. lateralis genome.

A型行为核心特征与血管紧张素转换酶基因型相关性研究

In order to study the role of inherited factors and Type A Behavior Pattern (TABP) in the development of CHD, the present study chose the angiotensin I-converting enzyme (ACE) gene as the target gene, and investigated the associations of TABP, the polymorphism of ACE gene with susceptibility to development of CHD in the healthy population and CHD patients from Northern China. 1. Correlation Analysis Between TABP and serum level of ACE in Chinese healthy individuals TABP and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and the serum level of ACE. 2 The distribution charicteristics of ACE gene polymorphism frequencies and association with serum level of ACE in Chinese healthy individuals population The polymorphism of ACE gene and serum of ACE were determined in 137 Chinese healthy individuals. The results showed that: the ethnic differences in I/D polymorphism of ACE gene are obvious; deletion polymorphism of the ACE gene is associated with serum ACE level. 3. The relationship between insertion/deletion polymorphism of ACE gene and CHD in a Chinese population I/D polymorphism in intron 16 of the ACE gene was determined by polymerase chain reaction(PCR) in a study of 109 patients with CHD. The results showed: The frequencies of DD genotype(0.39) and D allele(0.63) were higher among the CHD group than among the control subjects(0.12 and 0.42 respectively, P < 0.01). Furthermore, MI and multivessel disease was more strongly associated with (P < 0.01). It is indicated that D allele and DD gentype of ACE might be an important risk factor for CHD, especially for MI or multivessel disease in Chinese population. 4. Correlation Analysis Between Type A Behavior Pattern and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 291 Chinese healthy individuals. The result showed that the higher frequency of rare D allele of an insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) gene was found in type A behavior individuals compared with type B behavior individuals in 291 healthy individuals; there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. It is suggested that the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience may be associated with heredity. 5. Correlation Analysis Between Angiotensin I-Converting Enzyme Gene Polymorphism, Type A Behavior Pattern and Coronary Heart Disease in Chinese The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in 109 patients with CHD. The results showed the development of coronary heart disease(CHD) is influenced mainly by the behavioral attributes of competitiveness, achievement striving, hostility, being irritated easily and impatience; the deletion polymorphism of ACE gene may be play a important role in the process of it. 6. Correlation Analysis Between Type A Behavior Pattern Core Components and the Polymorphism of ACE Gene The polymorphism of ACE gene and type A behavior pattern (TABP) survey were determined in1306 Chinese healthy individuals. The results showed that there was a significant correlation between the scores of CH in TABP invertory and DD genotype of the ACE gene. Furthermore, the behavioral attributes of hostility, being irritated easily and impatience may be associated with heredity. At the end of this research, in terms of theory, the research approaches of TABP and the factors influenced the relationship between TABP and CHD were explored and discussed. Furthermore, several new opinions were put forward.

Avaliação de um protocolo de extração de DNA genômico a partir de sangue total.

2009

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n = 11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.

The lack of associations between alleles at the hypoxia-Inducible factor 1A C1772T loci and responses to acute hypoxia.

Objective The aim of this study was to investigate the associations between alleles of the hypoxia-inducible factor 1A (HIF1A) C1772T polymorphism and several physiological responses to hypoxia, including the hypoxic ventilatory response (HVR), and serum erythropoietin (EPO), arterial oxygen saturation (Sao2), and acute mountain sickness (AMS) responses during 8 hours of exposure to normobaric hypoxia. Methods A total of 76 males participated in the study; 52 participants completed an 8-hour exposure to 12.7% oxygen, during which time Sao2, EPO concentrations, and AMS scores were measured, while 62 individuals took part in an HVR trial (in total 38 individuals completed both protocols). DNA was obtained from leukocytes, and a 346-bp fragment of the HIF1A gene containing the C1772T polymorphism was amplified using polymerase chain reaction. Fragments were sequenced to reveal individual genotypes, and the associations between HIF1A genotype and EPO, Sao2, AMS responses to hypoxia and HVR were examined. Results The magnitude of the hypoxic responses was highly variable between individuals. The increase in participants' EPO responses ranged from 89% to 388% of baseline values following hypoxia, while Sao2 values during the exposure ranged from 71% to 89%. The HVR ranged from −0.04 to +2.18 L · min−1 · Sao2%−1 among participants. No significant differences in EPO, Sao2, AMS, or HVR results were observed between the HIF1A CC genotype and the combined CT/TT genotype group. Conclusion In this study, the HIF1A C1772T polymorphism does not appear to influence EPO, Sao2, or AMS responses during acute hypoxic exposure, or the magnitude of the HVR.

A general model of error-prone PCR

Pritchard, L., Corne, D., Kell, D.B., Rowland, J. & Winson, M. (2005) A general model of error-prone PCR. Journal of Theoretical Biology 234, 497-509.

Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light

Chungui Lu, Olga A. Koroleva, John F. Farrar, Joe Gallagher, Chris J. Pollock, and A. Deri Tomos (2002). Rubisco small subunit, chlorophyll a/b-binding protein and sucrose : fructan-6-fructosyl transferase gene expression and sugar status in single barley leaf cells in situ. Cell type specificity and induction by light. Plant Physiology, 130 (3) pp.1335-1348 Sponsorship: BBSRC RAE2008

Diagnóstico Molecular na era da sequenciação de 3ª geração e da PCR digital

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Premature ovarian aging in mice deficient for Gpr3

After becoming competent for resuming meiosis, fully developed mammalian oocytes are maintained arrested in prophase I until ovulation is triggered by the luteotropin surge. Meiotic pause has been shown to depend critically on maintenance of cAMP level in the oocyte and was recently attributed to the constitutive Gs (the heterotrimeric GTP-binding protein that activates adenylyl cyclase) signaling activity of the G protein-coupled receptor GPR3. Here we show that mice deficient for Gpr3 are unexpectedly fertile but display progressive reduction in litter size despite stable age-independent alteration of meiotic pause. Detailed analysis of the phenotype confirms premature resumption of meiosis, in vivo, in about one-third of antral follicles from Gpr3-/- females, independently of their age. In contrast, in aging mice, absence of GPR3 leads to severe reduction of fertility, which manifests by production of an increasing number of nondeveloping early embryos upon spontaneous ovulation and massive amounts of fragmented oocytes after superovulation. Severe worsening of the phenotype in older animals points to an additional role of GPR3 related to protection (or rescue) of oocytes from aging. Gpr3-defective mice may constitute a relevant model of premature ovarian failure due to early oocyte aging.