RESUMO
"Chelex 100" chelating resin has been previously proposed for the rapid extraction of human DNA for polymerase chain reaction. Protocols are given for the rapid extraction of bacterial and viral DNA from cultures or clinical samples. The DNA samples obtained were suitable for use in polymerase chain reaction.
Assuntos
Quelantes , DNA Bacteriano/isolamento & purificação , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Reação em Cadeia da Polimerase/métodos , Amplificação de Genes , Técnicas In Vitro , Resinas Sintéticas , Streptococcus agalactiae/genéticaRESUMO
With both a classic DNA preparation protocol (including removal of paraffin wax and protein digestion) and a DNA extraction protocol with Chelex 100, the hepatitis B virus genome was searched for using the polymerase chain reaction (PCR) in 30 samples of paraffin wax embedded liver tissue from patients with chronic hepatitis. The classic protocol was more sensitive than the rapid Chelex 100 procedure (10 v six positive samples). A third protocol, including removal of paraffin wax, protein digestion, and Chelex 100 treatment of the digestion solution before PCR, was more sensitive than the others (16 positive samples). It is concluded that it could therefore be helpful for PCR analysis of paraffin wax embedded liver tissue.
Assuntos
DNA Viral/análise , Vírus da Hepatite B/isolamento & purificação , Hepatite B/diagnóstico , Fígado/microbiologia , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Quelantes , Primers do DNA/química , Humanos , Dados de Sequência Molecular , Inclusão em Parafina , Resinas SintéticasRESUMO
We propose an one-step DNA extraction method suitable for the polymerase chain reaction. This procedure utilizes Chelex 100, a chelating in exchange resin. This technique was compared with a traditional technique (proteinase K lysis, phenol-chloroform extraction and ethanol precipitation) for isolation of human cytomegalovirus DNA from clinical samples. The procedure using Chelex 100 appeared to be a simple and fast extraction method for human cytomegalovirus DNA.
Assuntos
Infecções por Citomegalovirus/diagnóstico , DNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase , Líquido da Lavagem Broncoalveolar/microbiologia , Humanos , Leucócitos/microbiologia , Saliva/microbiologia , Urina/microbiologiaRESUMO
We describe a new protocol for extraction of DNA suitable for HIV1 gene amplification from clinical samples using "Chelex-100" chelating resin. Comparison was made with the classic proteinase K extraction method; 154 specimens were extracted with both methods and subjected to PCR (polymerase chain reaction). The Chelex-100 procedure optimized the yield of DNA recovery and minimized contamination due to sample manipulation. It decreased false negative results due to PCR inhibitors. A DNA sample suitable for use in PCR was obtained in 30 minutes. Chelex-100 treatment is a simple, rapid and low-cost method for DNA extraction in clinical laboratories.