SABIIA - Sistema Aberto e Integrado de Informação em Agricultura (Sabiia)

A simple and efficient method for extraction of Taq DNA polymerase

Murtaza,Naveed.

Amplified fragmentlength polymorphism (AFLP) markers have been used to ascertain the intensity of inherent diversity and relatedness in cotton (Gossypium spp.) plants. The effectiveness of this method to distinguish inter and intra specific difference in cotton could be handy in cultivar recognition and in marker assisted parental selection tool for plant breeders. Twenty cotton cultivars belonging to Gossypium hirsutum L., and G. arborium L. from the Pakistan and US origin were used for AFLP based genetic diversity estimates. The objective of this study was to assess the level of genetic variation among some cotton cultivars belonging to the old and new world species of cotton. Four EcoRI-MseI primer-pair combinations were used forthe AFLP analysis. The...

Tipo: Journal article

Palavras-chave: AFLP; Cluster analysis; Cotton; Genetic diversity; PCR.

Ano: 2006

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000400013

PCR-based sensitive detection of the edible fungus Boletus edulis from rDNA ITS sequences

Chen,Sique; Zheng,Xiujuan; Cao,Hongrui; Jiang,Linghui; Liu,Fangqian; Sun,Xinli.

Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with...

Tipo: Journal article

Palavras-chave: Ethanol precipitation; PCR; Purification; Taq DNA polymerase.

Ano: 2015

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000500005

Simple method to prepare DNA templates from a slice of peanut cotyledonary tissue for Polymerase Chain Reaction

Lian,Bin; Zang,Jin-ping; Hou,Wei-guo; Yuan,Sheng; Smith,Donald L.

Identification of commercially important fungi, such as the valuable edible fungus Boletus edulis can be difficult considering visual or metabolic approaches. Based on phylogenetic analysis of the rDNA ITS sequence, a pair of specific primers was designed for differentiating B. edulis from other mushrooms by PCR. PCR was performed with total DNA as a template at an annealing temperature between 56-60ºC. Positive amplicons were obtained from B. edulis with all DNA templates from fruit bodies and cultured mycelium, but not from other fungal species at an annealing temperature of 60ºC. The result indicated that B. edulis could be clearly distinguished from other fungi by PCR, and there were no misidentifications under the reaction conditions used. The primers...

Tipo: Journal article

Palavras-chave: Boletus edulis; Detection; Edible fungi; Internal transcribed spacer; PCR; Specific primers.

Ano: 2008

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000300011

A rapid and cheap protocol for preparation of PCR templates in peanut

Yu,Shu Tao; Wang,Chuan Tang; Yu,Shan Lin; Wang,Xiu Zhen; Tang,Yue Yi; Chen,Dian Xu; Zhang,Jian Cheng.

An efficient DNA extraction method was developed for peanut seed, where only 3-5 mg cotyledonary tissue was enough for more than 50 PCR reactions with a reaction volume of 15 μl. Both low copy number and high copy number DNA sequences were successfully amplified. Processing one seed sample only took about half an hour. Sampling had no significant effects on germination and development. The DNA extraction method makes it possible to identify transformants and conduct molecular marker studies prior to sowing, and thus may greatly hasten research progress.

Tipo: Journal article

Palavras-chave: Cotyledonary tissue; DNA extraction; Groundnut; PCR; Peanut.

Ano: 2010

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582010000400012

The lift pool method for isolation of cDNA clones from lambda phage libraries

Wang,Chuan Tang; Wang,Xiu Zhen; Tang,Yue Yi; Zhang,Jian Cheng; Yu,Shan Lin; Xu,Jian Zhi; Bao,Zhen Min.

This paper describes a simple, low cost and reliable DNA template preparation protocol for polymerase chain reaction (PCR) using immature leaves from peanut seeds or leaves from field-grown plants. The technique may find wide utility in studies involving PCR-based molecular markers, rapid screening for transformants and gene cloning.

Tipo: Journal article

Palavras-chave: DNA extraction; Groundnut; PCR; Peanut.

Ano: 2009

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000200009

Discrimination of Shark species by simple PCR of 5S rDNA repeats

LeBlanc-Straceski,Janine; Sobrado,Pablo; Betz,Sharon; Zerfas,Julie; Morgan,Karen.

A PCR based strategy was developed to screen a Xenopus oocyte λgt10 cDNA library. The PCR-based lift pool (LP) method follows the same two tiered strategy as conventional screening of phage libraries by filter hybridization. Two rounds of plating, one at high density to detect the clone, and one at low density to purify the clone to homogeneity, are performed. In the first round, lysates from high density plates, termed plate pools (PP), serve as template for PCR. In the second round, phage particles adhering to plaque lifts of low density plates are washed off nitrocellulose filters to create LPs, which are used as template for PCR. The integrity of the plaques on the low-density plates is preserved. Once a positive LP has been identified,...

Tipo: Journal article

Palavras-chave: CDNA; Lambda; Non-radioactive; PCR; Plaque; Screening.

Ano: 2006

URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000400012

Pinhal,Danillo; Gadig,Otto BF; Wasko,Adriane P; Oliveira,Claudio; Ron,Ernesto; Foresti,Fausto; Martins,Cesar.

Sharks are suffering from intensive exploitation by worldwide fisheries leading to a severe decline in several populations in the last decades. The lack of biological data on a species-specific basis, associated with a k-strategist life history make it difficult to correctly manage and conserve these animals. The aim of the present study was to develop a DNA-based procedure to discriminate shark species by means of a rapid, low cost and easily applicable PCR analysis based on 5S rDNA repeat units amplification, in order to contribute conservation management of these animals. The generated agarose electrophoresis band patterns allowed to unequivocally distinguish eight shark species. The data showed for the first time that a simple PCR is able to...

Distribution and conservation of the transposable element gypsy in drosophilid species

Palavras-chave: Chondrichthyes; PCR; Species identification; 5S rDNA; Sharks.

Ano: 2008

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572008000200033

Herédia,Fabiana; Loreto,Elgion Lúcio S.; Valente,Vera Lúcia S..

In an attempt to understand the dynamics of transposable elements (T'S) in the genome of host species, we investigated the distribution, representativeness and conservation of DNA sequences homologous to the Drosophila melanogaster gypsy retrotransposon in 42 drosophilid species. Our results extended the knowledge about the wide distribution of gypsy in the genus Drosophila, including several Neotropical species not previously studied. The gypsy-like sequences showed high divergence compared to the D. melanogaster gypsy element. Furthermore, the conservation of the restriction sites between gypsy sequences from phylogenetically unrelated species pointed to a more complex evolutionary picture, which includes the possibility of the horizontal transfer events...

Isolation and sequencing of seven Sox genes from the lacertid lizard Eremias breuchleyi

Palavras-chave: Drosophila; Retrotransposon; Gypsy; Southern blot; PCR.

Ano: 2007

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572007000100023

Jie,Li; Zheng,Ping-Ping; Song,Jiao-Lian; Rui,Jin-Long; Nie,Liu-Wang.

The Sox family of genes shares a high sequence similarity with the HMG box region of the human Y chromosomal gene, SRY. We used highly degenerate primers to clone and sequence seven Eremias breuchleyi Sox genes (EbSox2, EbSox3, EbSox4, EbSox11, EbSox12, EbSox14 and EbSox21). A database search for the cloned sequences revealed the following percentage identity with the homologous human SOX genes: EbSox2 = 96%, EbSox3 = 88%, EbSox4 = 94%, EbSox11 = 99%, EbSox12 = 96%, EbSox14 = 98%, EbSox21 = 97%. Cluster analysis indicates that they seem to belong to group B and group C of Sox gene family, respectively.

Medium-sized deletion in the BRCA1 gene: limitations of Sanger sequencing and MLPA analyses

Palavras-chave: Eremias breuchleyi; PCR; Sequence analysis; Sox genes; SSCP.

Ano: 2006

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572006000300031

Herman,Sandra; Varga,Dominic; Deissler,Heidrun L.; Kreienberg,Rolf; Deissler,Helmut.

We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized...

Use of the TSPY gene for sexing cattle

Palavras-chave: Mutation analysis; DNA sequencing; PCR; Hereditary breast cancer; Model calculations.

Ano: 2012

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572012000100007

Lemos,Daniela Cristina; Rios,Álvaro Fabrício Lopes; Caetano,Lisandra Cristina; Lôbo,Raysildo Barbosa; Vila,Reginaldo Aparecido; Martelli,Lúcia; Takeuchi,Paula Lumy; Ramos,Ester Silveira.

The Y-encoded, testis-specific protein (TSPY) is a Y-specific gene. The copy numbers of TSPY range from 20 to 60 in men and up to 200 in bulls. In this study, we examined the possibility of using the TSPY gene to sex cattle. DNA from blood samples of 100 Nelore cattle (50 males and 50 females) from the Nelore Cattle Breeding Program (PMGRN) was screened for TSPY by PCR using TSPY-specific primers. The assay was highly specific since all male samples were TSPY-positive and all female samples were negative. Positive results were also obtained at low DNA concentrations (less than 1 rhog/muL). These results showed that TSPY was a good male-specific marker, the usefulness of which was enhanced by the high copy number of the gene. This is the first report to...

Parentage testing and effect of misidentification on the estimation of breeding value in Gir cattle

Palavras-chave: Bovine; Cattle; PCR; Sexing; TSPY.

Ano: 2005

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572005000100020

Baron,Erica E.; Martinez,Mário L.; Verneque,Rui S.; Coutinho,Luiz L..

A correct relationship among sires is essential for an efficient breeding program. Microsatellite markers were used in progeny tests, to assess the paternity of seventy-four probable offspring of nine Gir dairy sires. A 36% misidentification rate was observed; however, these errors had minimal effects on the ranking of the nine bulls with regard to their genetic values. The results suggest that paternity tests should be performed in breeding programs, in order to prevent inappropriate paternities from influencing the genetic value of bulls in the future.

Association of MYF5 gene allelic variants with production traits in pigs

Palavras-chave: Microsatellite; Gir; Cattle; Breeding; Parentage; PCR.

Ano: 2002

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572002000400006

Carmo,Fausto Moreira da Silva; Guimarães,Simone Eliza Facioni; Lopes,Paulo Sávio; Pires,Aldrin Vieira; Guimarães,Marta Fonseca Martins; Silva,Marcos Vinícius Gualberto Barbosa da; Schierholt,Alex Sandro; Silva,Kleibe de Moraes e; Gomide,Lúcio Alberto de Miranda.

We studied the phenotypic effects of polymorphisms at the MYF5 gene in a divergent F2 swine population and found that one polymorphism was due to an insertion and another to a deletion. The genotypes of 359 F2 animals were obtained and the Normal/Normal (NN) and Normal/Insertion (NI) genotypes analyzed to determine associations with phenotypic data for performance, carcass and meat quality traits. Significant differences were observed (p < 0.05) between NN and NI animals for drip (NN = 3.14 ± 1.56; NI = 3.69 ± 2.78%), cooking (NN = 32.26 ± 2.41; NI = 33.21 ± 2.31%) and total loss (NN = 34.16 ± 2.63 and NI = 34.97 ± 2.08%). The Deletion marker was not statistically tested. The results indicate that the allelic variant Insertion is associated with a...

Variation in extragenic repetitive DNA sequences in Pseudomonas syringae and potential use of modified REP primers in the identification of closely related isolates

Palavras-chave: Myogenesis; DNA sequencing; Pig production; PCR.

Ano: 2005

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572005000300004

Çepni,Elif; Gürel,Filiz.

In this study, Pseudomonas syringe pathovars isolated from olive, tomato and bean were identified by species-specific PCR and their genetic diversity was assessed by repetitive extragenic palindromic (REP)-PCR. Reverse universal primers for REP-PCR were designed by using the bases of A, T, G or C at the positions of 1, 4 and 11 to identify additional polymorphism in the banding patterns. Binding of the primers to different annealing sites in the genome revealed additional fingerprint patterns in eight isolates of P. savastanoi pv. savastanoi and two isolates of P. syringae pv. tomato. The use of four different bases in the primer sequences did not affect the PCR reproducibility and was very efficient in revealing intra-pathovar diversity, particularly in...

Specific genotyping of human leukocyte antigen-A*01 by polymerase chain reaction using allele group-specific primers

Palavras-chave: Bacterial identification; Biodiversity; PCR.

Ano: 2012

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572012000400016

Kasuga,Ikuma; Paré,Peter D.; Sandford,Andrew J..

We established a specific genotyping assay for HLA-A*01, which is one of the most frequently found HLA-A alleles in the Caucasian population. This assay uses the polymerase chain reaction (PCR) with allele group-specific primers (ASP). HLA-A*01 group-specific primers were designed for exon 3 of the HLA-A gene, based on the recent HLA-sequence alignment. Both sense and anti-sense primers were designed with completely matched sequences to each specific HLA-A*01 allele, but mismatched by at least 1 nucleotide to all other known class I HLA alleles. By the use of these primers and stringent PCR conditions, we successfully genotyped the HLA-A*01 group alleles and achieved greater accuracy than previous methods.

Palavras-chave: HLA-A1; PCR; Allele group-specific primers.

Ano: 2006

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572006000200003

Trarbach,Ericka Barbosa; Monlleo,Isabella Lopes; Porciuncula,Carlos Guilherme Gaelzer; Fontes,Marshall Italo Barros; Baptista,Maria Teresa Mathias; Hackel,Christine.

Mutations in the KAL-1 gene localized at Xp22.3 have been shown to be responsible for the X-linked Kallmann syndrome (KS), a disorder characterized by the association of hypogonadotropic hypogonadism and anosmia. In this paper, we describe the investigation of two families with X-linked KS, in which similar interstitial deletions ning exons 5 to 10 of the KAL-1 gene were identified. The presence of interspersed repetitive DNA sequences within the KAL-1 gene might have predisposed to this type of mutation.

Tipo: Info:eu-repo/semantics/other

Palavras-chave: X-linked KS; Intragenic deletions; KAL-1 gene; PCR; Phenotypic variability.

Ano: 2004

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572004000300006

Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

Mirol,Patricia M.; Peral García,P.; Dulout,F.N..

A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eigth variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the...

PCR-based VNTR core sequence analysis for inferring genetic diversity in the shrimp Litopenaeus vannamei

Palavras-chave: MTDNA; Equine; PCR.

Ano: 2002

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572002000100006

Freitas,Patrícia Domingues de; Galetti Junior,Pedro Manoel.

The genetic variation in two farmed strains (F3-Panama and F17-Venezuela) of the shrimp Litopenaeus vannamei was examined based on DNA multiloci analyses. Eighteen adults of each strain were analyzed by PCR using a set of VNTR core sequence primers. Genetic similarity, mean allele frequency, mean heterozygosity and the frequency of polymorphic loci were determined for both strains. A dendrogram of genetic similarity was produced by UPGMA clustering. The results for three primers (INS, M13, YN73) revealed different levels of genetic variation within the strains. The higher genetic similarity seen within strain F17 was apparently related to inbreeding, although a bottleneck effect could not be discarded. The low level of genetic variability of this strain...

An analysis of multiplex-PCR in the detection of BCR-ABL transcripts in hematological disorders

Palavras-chave: Litopenaeus vannamei; Genetic diversity; Shrimp; VNTR; PCR.

Ano: 2002

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572002000400012

Sastre,Darío A.; Argaraña,Carlos E.; Heller,Viviana B.; Gallo,Mónica; Fernández,Enrique N.; Rodríguez,Cecilia M..

In this work, we describe the advantages of multiplex-PCR in the specific detection of BCR-ABL transcripts in different hematological disorders and its sensitivity compared to nested PCR. Fifty-three patients were studied for the presence of BCR-ABL transcripts: 24 patients with chronic myeloid leukemia (CML), 20 with acute leukemia (AL), and 9 patients with other hematological disorders. A variant rearrangement (b3a3) was found in a single case of CML (4.2%). Four out of the 20 patients with AL (20.0%) (14 adults, 6 children) were bcr-abl(+), and in this group three cases were classified as B-acute lymphoblastic leukemia (B-ALL), and one as acute myeloblastic leukemia (AML). Two of the three patients with B-ALL were positive for b2a2 and the other one for...

Tipo: Info:eu-repo/semantics/other

Palavras-chave: Multiplex-PCR; Ph chromosome; BCR-ABL; Chronic myeloid leukemia; PCR.

Ano: 2007

URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572007000400003

Identificação dos citoplasmas "S", "T" e "N" via PCR em populações de cebola no Vale do São Francisco

Horticultura Brasileira

Santos,Carlos Antonio F; Leite,Daniela L; Costa,Nivaldo D; Oliveira,Valter R; Santos,Ierla Carla N dos; Rodrigues,Marciene A.

A identificação do tipo de citoplasma em cebola foi facilitada com a publicação de primers de DNA específicos para os tipos "S", "T" e "N". O objetivo do presente trabalho foi identificar, por meio de marcadores moleculares, o tipo de citoplasma presente na cultivar de cebola BRS São Francisco e numa população experimental em desenvolvimento na Embrapa Semi-Árido, de forma a orientar o desenvolvimento de híbridos de cebola adaptados à região. Na amostra de 19 plantas da 'BRS Alfa São Francisco' observou-se a amplificação de fragmentos de 180 pb, ausência do fragmento de 414 pb com os primers 5'cob e a amplificação do fragmento de 473 pb dos primers OrfA501, indicando que o citoplasma presente nesta população é o citoplasma T. Foi também identificada a...