Comparison of methods for isolating high quality genomic DNA and total RNA from the mycelia of an oil palm pathogen, ganoderma boninense

L. I. Irene, Farah Diba Abu Bakar, A. S. Idris, Abdul Munir Abd. Murad

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Isolation of high quality and intact nucleic acids are important steps for a number of molecular techniques. The problems that are normally associated with nucleic acid isolation from filamentous fungi are the presence of high polysaccharide contaminants and tough cell walls. In this study, we compared several published protocols for efficient DNA and RNA isolation from the mycelia of an oil palm pathogen, Ganoderma boninense. Three protocols were analysed for the isolation of genomic DNA, with the best protocol being the hexacetyltrimethylammonium bromide (CTAB) method. This method yielded 336.5± 56.9 μg/ml genomic DNA from 0.1 gm of mycelia, with minimal carbohydrate contamination. This method also produced DNA of sufficient quality for PCR amplification of G. boninense DNA. A total of four protocols were analysed for RNA extraction. Among the four protocols, LiCl, SDS and phenol yielded the highest amount of total RNA, wherein a total of 359.83 ± 67.8 μg/ml total RNA from 0.1 gm of mycelia was obtained. Spectrophotometric assessment of the RNA indicated relatively high purity and an absence of carbohydrate contamination. This method produced RNA of sufficient quality that was suitable for RT-PCR of G. boninense genes.

Original languageEnglish
Pages (from-to)19-24
Number of pages6
JournalMalaysian Applied Biology
Volume44
Issue number1
Publication statusPublished - 2015

Fingerprint

Ganoderma
Mycelium
Elaeis guineensis
mycelium
Oils
RNA
genomics
pathogens
DNA
Nucleic Acids
nucleic acids
methodology
Carbohydrates
carbohydrates
Polymerase Chain Reaction
Phenol
bromides
Bromides
purity
Cell Wall

Keywords

  • Ganoderma boninense
  • Genomic DNA
  • Isolation
  • Protocol
  • Total RNA

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

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abstract = "Isolation of high quality and intact nucleic acids are important steps for a number of molecular techniques. The problems that are normally associated with nucleic acid isolation from filamentous fungi are the presence of high polysaccharide contaminants and tough cell walls. In this study, we compared several published protocols for efficient DNA and RNA isolation from the mycelia of an oil palm pathogen, Ganoderma boninense. Three protocols were analysed for the isolation of genomic DNA, with the best protocol being the hexacetyltrimethylammonium bromide (CTAB) method. This method yielded 336.5± 56.9 μg/ml genomic DNA from 0.1 gm of mycelia, with minimal carbohydrate contamination. This method also produced DNA of sufficient quality for PCR amplification of G. boninense DNA. A total of four protocols were analysed for RNA extraction. Among the four protocols, LiCl, SDS and phenol yielded the highest amount of total RNA, wherein a total of 359.83 ± 67.8 μg/ml total RNA from 0.1 gm of mycelia was obtained. Spectrophotometric assessment of the RNA indicated relatively high purity and an absence of carbohydrate contamination. This method produced RNA of sufficient quality that was suitable for RT-PCR of G. boninense genes.",
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AU - Abd. Murad, Abdul Munir

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