Comparação entre métodos de estocagem de DNA extraído de amostras de sangue, sêmen e pêlos e entre técnicas de extração

Coelho, E. G. A.; Oliveira, Denise Aparecida Andrade de; Teixeira, Cristina Mara; Sampaio, Ivan Barbosa Machado; Rodrigues, Salatir; Alves, Ceres Luciana

doi:10.1590/s0102-09352004000100017

Cited by 10 publications

(15 citation statements)

References 4 publications

(1 reference statement)

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“…DNA extraction was carried out at the Laboratory of Genetics in the Animal Science Department at the Veterinary School (LGEV) of the Federal University of Minas Gerais (UFMG), Brazil, according to the method described by Coelho et al (2004). PCR mixtures were prepared with 5.0 µL Phusion Flash Master Mix Enzyme, 1.0 µL ultrapure water, 3.0 µL primer mix, and 1.0 µL extracted DNA.…”

Section: Methodsmentioning

confidence: 99%

Genetic relationship between the Nordestino horse and national and international horse breeds

Pires

Coelho

Melo³

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Knowledge of genetic diversity and relationships between breeds is very important for conservation programs. Hair samples were collected from 393 individual Nordestino horses and genotyped using 14 microsatellite markers in order to investigate the genetic relationship between this breed and 66 international horse breeds. There was high allelic diversity and inbreeding coefficient within population values were not significant, which was probably due to crossbreeding. Despite the Nordestino horse population being in Hardy-Weinberg equilibrium, a global deficit of heterozygotes was observed. This may represent evidence of repeated use of the same stallions for breeding, which is consistent with the high number of castrated males found. Campolina, Mangalarga Marchador, and Mangalarga were the Brazilian horse breeds most closely related to the Nordestino horse, which is a reflection of recent introgressions. Among Iberian horse breeds, the Sorraia breed appears to have had an important influence on the genetics of the Nordestino horse. Those results provide important information that can guide future conservation programs.

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Section: Methodsmentioning

confidence: 99%

Genetic relationship between the Nordestino horse and national and international horse breeds

Pires

Coelho

Melo³

et al. 2016

Genet. Mol. Res.

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“…Thus, a good DNA extraction protocol should be fast, practical, cheap, free of contamination and toxicity, and produce DNA of high quantity and quality to be used in PCR (Barea et al, 2004;Aidar and Line, 2007) with minimal fragmentation (Yang et al, 2008). However, many conventional DNA extraction protocols do not have these characteristics (Goldenberger et al, 1995), thus limiting their usefulness for producing DNA templates for PCR (Coelho et al, 2004;Manuja et al, 2010). In the present study, we found that the wide use of the DNeasy Blood & Tissue Kit is not justified based on results of the spectrophotometric assay, which showed low DNA concentrations.…”

Section: Discussionmentioning

confidence: 99%

“…PCR is frequently used in clinical diagnostics (Masri et al, 1997;Manterola et al, 2003;Grom et al, 2006;Sharifzadeh et al, 2011), animal breeding programs (Lien et al, 1993;Coelho et al, 2004;Milazzotto et al, 2008), progeny tests, determination of sex ratios in samples where X-bearing and Y-bearing sperm are separated (Resende et al, 2009), and in forensic science (Hanson and Ballantyne, 2004;von Wurmb-Schwark et al, 2006;Koch and Andrade, 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Despite its wide applicability, the success of the PCR method depends on the quality and quantity of the DNA template (Coelho et al, 2004;von Wurmb-Schwark et al, 2006), which should be free of contaminants and DNA nucleases that impair the amplification process (Manuja et al, 2010). There are many methods currently available for DNA purification; however, problems related to contamination with foreign DNA, PCR inhibitors, and DNA susceptibility to fragmentation still persist (Coelho et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

“…There are many methods currently available for DNA purification; however, problems related to contamination with foreign DNA, PCR inhibitors, and DNA susceptibility to fragmentation still persist (Coelho et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis

Silva¹,

Pelinca²,

Acosta³

et al. 2014

Genet. Mol. Res.

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ABSTRACT. Successful DNA extraction is indispensable for molecular methods based on polymerase chain reaction (PCR); however, goat sperm DNA extraction is limited. Thus, the aim of this study was to evaluate three methods to extract DNA from goat sperm for use in PCR. Eight goat semen pools were used for DNA extraction by using the DNeasy Blood & Tissue Kit, phenol-chloroform, and Chelex-100 methods. DNA samples were analyzed spectrophotometrically to determine the DNA concentration and purity, visualized on 0.8% agarose gel, and used at different amounts (150, 100, 50, 10, and 1 ng) for PCR with electrophoresis, followed by 1.5% agarose gel electrophoresis. The quantity of DNA extracted with Chelex-100 was DNA extraction from goat sperm for PCR analysis higher (P < 0.05) than that obtained with either the DNeasy Blood & Tissue Kit or the phenol-chloroform method, with the phenolchloroform method yielding a greater quantity (P < 0.05) than the kit. The DNeasy Blood & Tissue Kit produced a higher (P < 0.05) purity product than the Chelex-100 method, and all samples obtained by the three protocols were positive for DNA, as assessed by electrophoresis. All of the different concentrations of DNA produced by these methods were amplified by PCR, although for DNA produced by the phenolchloroform method, PCR was only possible after complementary purification. In conclusion, the Chelex-100 method is cheap, secure, simple, fast, and effective, and is a potential tool for extracting goat sperm DNA without limitations in PCR.

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Comparative analysis of DNA extraction protocols in free-range chickens with respect to efficiency, extraction facility and cost

Bartholazzi¹,

Torres²,

Quirino³

et al. 2015

Pubvet

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Objetivou-se, com este trabalho testar três diferentes protocolos de extração de DNA, sendo dois para amostras de sangue e um para amostra de bulbo de penas, considerando a eficiência de extração, a quantidade, a qualidade do DNA extraído e o custo de extração. Foi coletado sangue de galinhas caipiras, através da punção da veia braquial e foram coletadas as penas da região peitoral em uma propriedade localizada no estado do Rio de Janeiro. Os protocolos testados foram: 1) Extração alcalina rápida pela coleta de sangue total; 2) Extração alcalina rápida do bulbo das penas da região peitoral e 3) Extração de DNA com kit comercial (Nucleo Spin® Tissue) para sangue em FTA Card (NucleoCard). Em todos os protocolos, após a extração, as amostras foram quantificadas e verificadas a concentração e a qualidade do DNA, através de espectrofotometria. Constatou-se que os protocolos 1 e 2 apresentaram bons resultados na quantidade do DNA extraído, porém contatou-se alta concentração de proteínas. No protocolo 3, observou-se baixa quantidade de DNA extraído, mas apresentando maior pureza. Todos os protocolos mostraram-se satisfatórios quanto à qualidade na amplificação, entretanto, pode-se concluir que os protocolos de extração alcalina (1 e 2)apresentaram maior facilidade de uso, rapidez na extração e menor custo quando comparados ao protocolo 3.

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Comparação entre métodos de estocagem de DNA extraído de amostras de sangue, sêmen e pêlos e entre técnicas de extração

Cited by 10 publications

References 4 publications

Genetic relationship between the Nordestino horse and national and international horse breeds

Genetic relationship between the Nordestino horse and national and international horse breeds

Comparative study of DNA extraction methodologies from goat sperm and its effects on polymerase chain reaction analysis

Comparative analysis of DNA extraction protocols in free-range chickens with respect to efficiency, extraction facility and cost

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