Download T. Hill

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
(general DNA fluorophore or specific probe)
Light output from PCR reaction
•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
54,000
1,000,000
0
5
10
100,000
10,000
15
PCR cycle number
1,000
20
100
25
30
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
Probe
Probe is
both a PCR
primer and
probe
Fluorophore
PCR primer
Quencher
Primer part of
Scorpion binds to
16S rRNA gene
Taq polymerase copies the single stranded DNA
Heat drives off lower DNA strand
Probe binds to target,
fluorophore held apart from quencher,
and light is emitted
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)