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Multiple Displacement Technology Goes Digital: a method for enumeration of bacterial nucleic acid contamination in enzymes

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Is bacterial DNA contamination in your whole genome amplification kit a problem? For microbiologists it sure is. WGA is a technique where the complete genomic content of a sample is amplified non-specifically and at a single temperature (isothermically). The presence of any contaminating DNA in the enzyme or buffers will not only use up the reaction components, but add miscellaneous sequences to the real sample.

Many microbiologists are attempting to ask questions using single-cell genomics but the presence of contamination in enzyme kits makes this difficult. That was why scientists at Stanford University, Dr. Stephen Quake and Dr. Paul Blainey set out to express and purify their own DNA-free phi29 polymerase for use in whole genome amplification. And in the process, they figured out how to perform single-cell genomics without the bias introduced by WGA.

Thanks to their experience with digital PCR, a technique that allows for sensitive low level quantification of gene targets by diluting down the template until there is only one DNA per PCR reaction, they were able to take this to the next level. They combined digital amplification technique with whole genome amplification and designed an assay that could detect DNA contamination down to 1 fg/ ml. Compare that to ng/ml for digital PCR and you can see how advantageous this method can be.

Using their high purity Phi29 DNA polymerase that was shown to be DNA-free, they can now perform single-cell genomics experiments and be sure that the DNA they amplify is their target and nothing else.

This work is summarized in the blog article linked above and the paper, called “Digital MDA for enumeration of total nucleic acid contamination” , was published in Nucleic Acids Research Advance Access November 11, 2010
 
 

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