Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR)

Barea, Jaqueline A.; Pardini, Maria Inês de Moura Campos; Gushiken, Tsieko

doi:10.1590/s1516-84842004000400008

Cited by 25 publications

(23 citation statements)

References 21 publications

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“…However, there was no PCR amplification of DNA extracted using the phenol-chloroform protocol for all DNA concentrations used unless an additional purification step with chloroform-isoamyl alcohol was performed. This fact was also observed by Barea et al (2004), who showed the generation of PCR inhibitory substances by organic solvents. Moreover, the use of a low SDS detergent concentration (0.001%) in the phenol-chloroform procedure may act as an inhibitor of Taq polymerase in PCR (Goldenberger et al, 1995).…”

Section: Discussionsupporting

confidence: 72%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, Chelex-100 chelates polyvalent metal ions, which may act in the breakdown of DNA and PCR inhibition (Sepp et al, 1994;Barea et al, 2004). As this protocol only requires a low sample volume (25 μL) (Manuja et al, 2006), it can be applicable to situations where the quantity of biological material is limited, for example, in forensic assays (Koch and Andrade, 2008;Walsh et al, 1991).…”

Section: Discussionmentioning

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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Section: Discussionsupporting

confidence: 72%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…When leukocytes and plasma were analyzed separately, this factor was eliminated ( 14) (47) (48) . In these samples, STNPCR had higher sensitivity than conventional nested PCR using whole blood (7) (13) (39) .…”

Section: Discussionmentioning

confidence: 99%

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Lima

Guedes

Lima

et al. 2015

Rev. Soc. Bras. Med. Trop.

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Introduction: Molecular analyses are auxiliary tools for detecting Koch's bacilli in clinical specimens from patients with suspected tuberculosis (TB). However, there are still no effi cient diagnostic tests that combine high sensitivity and specifi city and yield rapid results in the detection of TB. This study evaluated single-tube nested polymerase chain reaction (STNPCR) as a molecular diagnostic test with low risk of cross contamination for detecting Mycobacterium tuberculosis in clinical samples. Methods: Mycobacterium tuberculosis deoxyribonucleic acid (DNA) was detected in blood and urine samples by STNPCR followed by agarose gel electrophoresis. In this system, reaction tubes were not opened between the two stages of PCR (simple and nested). Results: STNPCR demonstrated good accuracy in clinical samples with no cross contamination between microtubes. Sensitivity in blood and urine, analyzed in parallel, was 35%-62% for pulmonary and 41%-72% for extrapulmonary TB. The specifi city of STNPCR was 100% in most analyses, depending on the type of clinical sample (blood or urine) and clinical form of disease (pulmonary or extrapulmonary). Conclusions: STNPCR was effective in detecting TB, especially the extrapulmonary form for which sensitivity was higher, and had the advantage of less invasive sample collection from patients for whom a spontaneous sputum sample was unavailable. With low risk of cross contamination, the STNPCR can be used as an adjunct to conventional methods for diagnosing TB.

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“…The customary quantification of extracted DNA by spectrophotometry, was not undertaken in our study because sequence amplification by PCR is apparently most influenced by the quality of the DNA extracted from fixed tissues (Shi et al, 2002;Barea et al, 2004).…”

Section: Scatena and Morielle-versute 163mentioning

confidence: 99%

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

Scatena

Morielle‐Versute

2008

Genet. Mol. Biol.

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To establish a technique which minimized the effects of fixation on the extraction of DNA from formalin-fixed tissues preserved in scientific collections we extracted DNA samples from fixed tissues using different methods and evaluated the effect of the different procedures on PCR and sequencing analysis. We investigated muscle and liver tissues from museum specimens of five species of fruit-eating (frugivorous) bats of the Neotropical genus Artibeus (Chiroptera, Phyllostomidae): A. fimbriatus, A. lituratus, A. jamaicensis, A. obscurus, and A. planirostris. The results indicated that treatment of tissues in buffered solutions at neutral pH and about 37°C for at least four days improves the quality and quantity of extracted DNA and the quality of the amplification and sequencing products. However, the comparison between the performance of DNA obtained from fixed and fresh tissues showed that, in spite of the fact that both types of tissue generate reliable sequences for use in phylogenetic analyses, DNA samples from fixed tissues presented a larger rate of errors in the different stages of the study. These results suggest that DNA extracted from formalin-fixed tissue can be used in molecular studies of Neotropical Artibeus bats and that our methodology may be applicable to other animal groups.

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Extração de DNA de materiais de arquivo e fontes escassas para utilização em reação de polimerização em cadeia (PCR)

Cited by 25 publications

References 21 publications

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

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

Single-tube nested PCR assay with in-house DNA extraction for Mycobacterium tuberculosis detection in blood and urine

Suitability of DNA extracted from archival specimens of fruit-eating bats of the genus Artibeus (Chiroptera, Phyllostomidae) for polymerase chain reaction and sequencing analysis

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