DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London.

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Presentation transcript:

DNA-based Methods for Quantifying Microbes in Atmospheric Samples Tom Hill, Helen Ahern and Bruce Moffett University of East London

Quantitative PCR: theory qPCR exploits the amplifying power of the Polymerase Chain Reaction to count the number of genes in a sample It uses the number of cycles of amplification required to first detect the amplified gene: the more PCR cycles needed the fewer copies of the gene were present at the start

qPCR: theory Standards (red to yellow) with known numbers of copies of a gene are used to generate a standard curve The number of gene copies in an unknown (blue) can then be calculated 100,00010,000 1, ,000,000 54,000

qPCR: DNA fluorophores A non-specific DNA stain, such as Sybr Green I (which binds to double stranded DNA) can be used to make any PCR reaction quantitative Advantages: rapid start-up (ie, no probe design), cheap, high light output Disadvantages: false positives can result if the PCR reaction is not completely specific for the target gene Pseudomonas-specific qPCR (using Sybr Green I) of winter heath phyllosphere DNA. Sample (dotted line) contained 15,100 copies of the Pseudomonas 16S rRNA gene.

qPCR: DNA probes A light-emitting DNA probe can also be used for detection of the PCR product. Various probe designs exist but use the bringing together or separation of two fluorophores (when the DNA probe binds to the target) and exploit the transfer of fluorescence resonance energy between them (excitation of one fluorophore by another) Examples include TaqMan, Hyb-probe, molecular beacons and scorpion probes Advantages: highly-specific and reliable detection of the amplified target gene Disadvantages: design and optimisation needed, cost Shown over the next two slides is the action of a Scorpion probe we use to quantify ammonia-oxidizing bacteria

qPCR: potential uses Detection levels using qPCR can be very low (eg, down to 10 copies of a gene in a sample) if the primers amplify efficiently. Our primers for Pseudomonads and inaW show this potential. We will use them on cloud and phyllosphere samples to: quantify total bacteria (using universal bacterial 16S primers with DNA) quantify active bacteria (using universal bacterial 16S primers with RNA) quantify total Pseudomonads (using Ps-specific 16S primers with DNA) quantify active Pseudomonads (using Ps-specific 16S primers with RNA) quantify total IN genes (eg, using inaW-specific primers with DNA) quantify expressed IN genes (eg, using inaW-specific primers with RNA)