I want to isolate DNA from live bacteria only to analyse bacterial community dynamics. Has anybody experience in 'differential isolation' from live cells only. I have found Ultra-Deep Microbiome Prep might be a solution but it looks like quite a new thing. There is little info on it.
The Ultra-Deep Microbiome prep is ultimate solution for the removal of non-target host DNA elimination of DNA from dead microbes and extraction of DNA only from live bacteria. The Ultra-Deep Microbiome Prep Kit allows significantly deeper sequencing compared to conventional DNA isolation solutions. The Ultra-Deep Microbiome Prep is the perfect kit for ultra-sensitive identification of a broad range of bacteria from a variety of sample types. DNA extraction is supplied for whole blood, primary body fluids, including CSF and synovial fluid, BAL, exudates like sputum and urine, human and animal tissues, biopsies, and biofilms. As an important character the Ultra-Deep Microbiome Prep enables detecting and indentifying microorganisms that are viable but not growing in culture. Microbiome DNA enriched by the Ultra-Deep Microbiome Prep Kit is compatible to commercially available NGS library kits and sequencing kits
Very thanks for that description but did you ever used the kit, compared it with other in-lab developed techniques.
Does it yields pure live microbes DNA only or a mixture of 'native' DNA (from live microbes) and chemically-damaged host/dead microbes DNA. I am considering a scenario I will purify decent quantities of DNA (e.g. measured by A260, or even using fluorochrmoes) but in large extent inaccessible to polymerase.
I have been using the Ultra Deep Microbiome kit in an attempt to isolate bacteria from mouse lung tissue with some but limited success due to the properties of my lung tissue. All bacteria not in intact cells should be eliminated by this method due to the DNase treatment before bacterial lysis. For my purposes this is especially important for eliminating the mouse DNA which is much more prevalent in whole tissue lysis and effectively blocks analysis of the low biomass of bacteria present in the lung. Because of this it is also critical not to freeze samples before the initial steps of the protocol to prevent damage to less sturdy bacterial species skewing your results. One critical advantage of the UDM kit is the stringent care they have taken to eliminate contaminating DNA from the reagents and other kit components which is a real problem with low biomass bacterial samples. Most of the other commercial preps have significant bacterial DNA contamination in their enzymes and reagents so analysis of mock extractions is critical. It is also true that much of the microbiome work does not included a DNAse step to eliminate dead bacteria from the analysis and one that does (Pezzulo et al., Appl. Environ. Microbiol. 2013, 79(19):5936) finds mostly DNAse sensitive bacterial DNA in healthy pig lungs. This speaks to my reference to having only limited success because more than half of my samples from lungs of SPF mice living on ventilated air are negative for any bacterial 16S rDNA unless they have been previously innoculated. I would recommend the UDM kit as giving a much lower rate of false positives.
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