Wei sheng wu xue bao = Acta microbiologica Sinica · 2014
[Preparation of metagenomic DNA from bronchoalveolar lavage fluids of patients with chronic obstructive pulmonary diseases].
Аннотация на английскомСтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Wang Juan, Shen Ning, Du Yipeng, Erb-Downward John R, Huffnagle Gary B, Gyetko Margaret R, He Bei
Аннотация
OBJECTIVE: To optimize the method of isolating a small amount of metagenomic DNA efficiently from bronchoalveolar lavage fluids (BALF) of patients with stable chronic obstructive pulmonary diseases (COPD) , which will facilitate subsequent PCR and DNA sequencing.
METHODS: BALF (5mL) of stable COPD patients was spun down to collect the cells. To extract genomic DNA from Gram-positive bacteria more efficiently, QIAGEN's DNA extraction protocol was optimized as follows: Added Buffer ATL to the pellets and used bead tubes and tissue homogenizers to break cell walls; then added proteinase K and incubated; after adding Buffer AL and ethanol, pipetted the mixture into a DNeasy spin column then centrifuged; washed the column with Buffer AW1 and Buffer AW2, finally added 50 microL Buffer AE to elute DNA. After measuring the total DNA concentration, the bacterial 16S rDNA was amplified by PCR and amplicon libraries were created for further determination.
RESULTS: The DNA content of BALF with optimized protocols was 467.5 (135.0-1697.5) ng, which was significantly higher than those extracted with phenol-chloroform 95.0 (0-612.5) ng. After optimizing, more 16S rDNA PCR production can be obtained for future analysis (P = 0.002).
CONCLUSION: The optimized DNA extraction methods combining DNA isolation kits with bead-beating were more efficient in isolating tiny metagenomic DNA from BALF.
METHODS: BALF (5mL) of stable COPD patients was spun down to collect the cells. To extract genomic DNA from Gram-positive bacteria more efficiently, QIAGEN's DNA extraction protocol was optimized as follows: Added Buffer ATL to the pellets and used bead tubes and tissue homogenizers to break cell walls; then added proteinase K and incubated; after adding Buffer AL and ethanol, pipetted the mixture into a DNeasy spin column then centrifuged; washed the column with Buffer AW1 and Buffer AW2, finally added 50 microL Buffer AE to elute DNA. After measuring the total DNA concentration, the bacterial 16S rDNA was amplified by PCR and amplicon libraries were created for further determination.
RESULTS: The DNA content of BALF with optimized protocols was 467.5 (135.0-1697.5) ng, which was significantly higher than those extracted with phenol-chloroform 95.0 (0-612.5) ng. After optimizing, more 16S rDNA PCR production can be obtained for future analysis (P = 0.002).
CONCLUSION: The optimized DNA extraction methods combining DNA isolation kits with bead-beating were more efficient in isolating tiny metagenomic DNA from BALF.