1 Advanced Environmental Biotechnology II Week 07 - Quantitative PCR

2 This is based on Quantitative real-time PCR Cindy J.Smith chap 6 in Molecular Microbial Ecology BIOS Advanced Methods. (2005) Osborn, A. Mark.; Smith, Cindy J. Eds. Taylor & Francis Routledge

3 Molecular methods tells a lot about microbial diversity and function. Often depends on the polymerase chain reaction (PCR). Classical end-point PCR introduces biases as the mixed environmental target template is amplified.

4 The PCR amplicons cannot be used accurately to quantify numbers or proportions of specific genes or phylotypes within natural environments.

5 End-point PCR works by exponentially amplifying the target gene over a number of cycles to result in detectable levels of the gene. It assumes that all genes within a mixed community are amplified at equal efficiencies by the primer set used, and that the yields of specific products are not influenced by either PCR conditions or cycle number.

6 Initially in a PCR amplification, primers are present as an excess in proportion to the template concentration. At some point in the reaction the template concentration increases to a critical amount that makes it as likely to bind to itself rather than to anneal with the primers. This results in competition events between template-template and template-primer hybridizations. Therefore the amplification of that particular template is now self-limiting.

7 Meanwhile templates that were originally less abundant are still being exponentially amplified by the primers without self- inhibition. Theoretically this can result in equal end-point ratios of template from unequal starting template ratios.

8 Prior to the development of real- time PCR, the other strategies available for quantification were competitive PCR and limiting dilution PCR. These methods are both time- and resource- consuming and require post- PCR analysis.

9 Quantification of genes can be achieved using quantitative real-time PCR (qPCR). Data is collected as the genes are amplified in ‘real time’. Gene quantification is carried out during the initial cycles of PCR, before PCR kinetics bias the reaction. Since product yields at this stage are below the detection limits of ethidium bromide, fluorescence-based labeling is used, whereby a fluorescent dye is detected that accumulates in direct proportion to the yield of the amplified PCR product.

10 As a result, template amplification is recorded for every cycle via the corresponding increase in fluorescence. Quantification of the starting template is achieved by determining the threshold cycle (Ct) of the unknown samples and of a range of known standards. The Ct value is defined as the point at which the accumulation of amplicons, as measured by an increase in fluorescence, is significantly above the background levels of fluorescence. Yields during the very first cycles of amplification cannot be determined as fluorescence levels are indistinguishable from background levels.

11 Once the Ct value is exceeded, the exponential accumulation of product can be measured by the increase in fluorescence. Amplification is not affected by PCR drift as product renaturation is not competing with primer annealing. Amplified gene copy numbers are proportional to those of the initial template. The greater the initial template concentration, the earlier the Ct value is reached, whilst reactions containing lower concentrations of template will require a greater number of amplification cycles before the fluorescence level is greater than the background level.

Quantification Use standard curves constructed from known amounts of the target gene. Standard curves can be created from genomic DNA/RNA, plasmid DNA or from a PCR product of the target gene. The range of concentrations of the standard template DNA should span the expected amount of the unknown samples. The known standards should be amplified using the same PCR conditions as used for the unknown samples and Ct values are determined for each.

13 A linear regression line is constructed from the Ct values of the standards plotted against the log of their starting concentration. Unknown samples are then quantified by comparing their Ct values to the standard curve. Determination of Ct values, standard curve construction and unknown gene quantification is carried out using the software accompanying the realtime PCR system in use.

14 Quantification is dependent upon the reproducibility of both the real-time PCR system used and the experiment. Every aspect of the real-time PCR from pipetting of the standards and unknowns to the efficiency of the PCR amplification will influence reproducibility. The efficiency of the amplification of the standard and of the unknown environmental samples should be the same. However, environmental samples routinely contain inhibitory compounds that affect the efficiency of the PCR reaction. So, determine the optimal dilution of environmental template DNA that will reduce inhibition to a minimum and therefore increase the PCR efficiency.

15 10 ng DNA 1 ng DNA 100 pg DNA10 pg DNA 1 pg DNA no DNA

16 (A) Typical PCR amplification plot generated by real-time PCR. Cycle number is plotted against the fluorescence. Quantification of starting template by real-time PCR occurs during the linear amplification stage.

qPCR chemistries A number of different chemistries are available for quantification of template DNA. The following sections will review the two most widely used methods in the literature, namely the Taq nuclease assay (TNA) using TaqMan® probes (PE Applied Biosystems, Foster City, CA, USA) and SYBR Green detection.

Taq nuclease assay This method exploits the 5′ exonuclease activity of the polymerase enzyme used in combination with fluorescent resonance energy transfer (FRET) technology. An oligonucleotide is designed to anneal just downstream of the forward primer. This oligonucleotide, known as a TaqMan probe, consists of a fluorescent molecule attached to the 5′ end and a quencher molecule at the 3′ end.

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21 When the fluorescent molecule and the quencher are in close proximity the probe does not fluoresce due to the transfer of energy from the high-energy fluorescent molecule to the low-energy quencher (FRET). During the elongation step, the 5′ exonuclease activity of the Taq polymerase cleaves nucleotides from the probe molecule; as this occurs the fluorescent molecule bound to the 5′ end of the TaqMan probe is removed from the path of the newly forming amplicon. Once the fluorescent molecule and quencher are no longer in close proximity, fluorescence is emitted and can then be detected.

22 The fluorescence detected is therefore a direct measure of the amount of amplified target template. A major advantage of this method is that fluorescence is only generated by cleavage of the sequence-specific probe during amplification of the target template, and hence non-specific amplification products are not detected. However, design of primers and probes for TaqMan assay is restricted by the requirement of an additional conserved site necessary for probe hybridization.

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24 1.The PCR reaction is prepared as usual, and the reporter probe is added. 2.As the reaction starts both probe and primers anneal to the DNA target. 3.Polymerisation of a new DNA strand is begun from the primers, and once the polymerase reaches the probe, its 5'-3-exonuclease degrades the probe, separating the fluorescent reporter from the quencher, resulting in an increase in fluorescence. 4.Fluorescence is detected and measured and its increase is used to work out the amount of DNA.

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27 10 ng DNA 1 ng DNA 100 pg DNA10 pg DNA 1 pg DNA no DNA * * * * * *Cycle Threshold: Cycle # at which growth curve = 30 fluorescence units (significantly above background) A direct correlation between the starting number of target sequence copies and the number of PCR cycles needed to first see an increase in reporter dye fluorescence.

28 Step 1. Collect water sample

29 Step 2. Filter water sample

30 Step 7. Transfer sample to reaction tube containing PCR reagents

31 Step 8. Place reaction tube in real-time thermal cycler

32 Step 9. Run reaction in thermal cycler

33 Step 10. Import run data into spreadsheet and calculate target cells in sample _____________________________________________________________________________ Sample Entero Control dCT Calib. ddCT Ratio Calib. cells QPCR cells __________CT____CT____________dCT_________________________________________ 5A E __ 5B E C E __ 2A E __ 2B E __ 2C E __

SYBR green SYBR Green binds to double stranded DNA (dsDNA) and emits a fluorescent signal only when bound. By adding it to the qPCR reaction mixture it can be used to track the accumulation of amplicons in ‘real time’ at the end of each elongation stage of each PCR cycle when all amplicons are double-stranded. The increase in fluorescence emissions with every cycle can be translated into quantitative results by comparison with a standard curve.

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37 An advantage of using SYBR Green is that no additional probes are required for detection. However, SYBR Green will bind to any double-stranded DNA molecule in the reaction. This may be an issue if unspecific products and/or primer dimers are formed, as these will contribute to the overall fluorescence measured resulting in an overestimation of starting template. Non-specific product formation can be a particular problem when quantifying low copy numbers.

38 The presence of primer dimers and nonspecific target in an SYBR Green reaction can be identified by a melting curve (dissociation curve). A melting curve is created by measuring fluorescence during denaturation of the amplified product with increasing temperatures. The shorter primer dimers will dissociate first due to their lower melting temperature (Tm). The Tm of the product may also be used to identify whether or not the correct amplification product was generated, thus eliminating the need for gel electrophoresis.

Applications qPCR has been used to examine: - total microbial communities and the relative proportions of specific phylotypes within a number of different environments using the 16S rRNA gene. - targeting of functional genes

40 phylotypes (bacteria defined only by their 16S rDNA sequence)

qPCR analysis of total microbial community rRNA genes Aquatic microbial communities TaqMan probes Compared a number of primers and TaqMan probes for the domains Bacteria and Archaea also phylogenetic-specific primers and probes for Synechococcus and Prochlorococcus. Standard curves constructed from linearized plasmid Relative proportion of groups determined over a 200 m depth profile in seawater Then determined Synechococcus spp. to the overall cyanobacterial population in Lake Constance. Dynamics of Synechococcus populations and subpopulations to be followed over a 1 year period.

42 The SYBR Green method has been used to quantify Pseudoaltermonas species relative to total eubacterial abundance in a range of different marine habitats.

43 Aquatic environments are relatively free from compounds that inhibit the polymerase enzyme activity. Serial dilutions of DNA extracted from lake water did not indicate the presence of any inhibitory substances. However, the presence of inhibitors in soil and sediment extractions has been well documented.

44 qPCR used to quantify bacteria present in environments that contain compounds inhibitory to PCR, for example ammonia- oxidizing bacteria (AOB) in arable soil and in a municipal waste-water treatment plant. Archaeal abundance in a range of temperate environments such as different soil types, sediment and microbial mats and archaea inhabiting the extreme environments of hot spring water, surrounding anoxic sediment and hydrothermal vent effluent have also been quantified.

45 The effects of inhibitors on real-time PCR was examined using TaqMan qPCR to quantify Geobacter spp. present in aquifer sediment. Humic acids do reduce the activity of the polymerase but also the presence of inhibitors can additionally quench fluorescence, resulting in higher Ct values and an underestimation of the target gene. To reduce this effect, run a series of template dilutions for all environmental samples.

Functional ecology Functional roles of microorganisms in the environment can also be investigated. Primers and probes can be designed to target conserved sequences of a functional gene. The target can be either DNA to quantify gene abundance, or mRNA for gene expression studies. Standard curves are constructed using a target sequence.

47 If mRNA the standard curve should be constructed from cDNA and not double- stranded DNA, as otherwise there is no control for the efficiency of the RT reaction. curve constructed from: - a known amount of RNA that has been reverse-transcribed into cDNA and then serially diluted - a serial dilution of known quantities of RNA each of which are then individually reverse- transcribed into cDNA and used in the qPCR reaction to construct the standard curve.

48 Using DNA as the target, genes that play key roles in important biogeochemical cycles have been quantified using SYBR Green : denitrification processes have been investigated via the nitrite reductase gene nirS; methane oxidation by methanotrophs in soil via the pmoA gene; ammonium oxidation by AOB. Other quantification of key genes involved the biodegradation of aromatic pollutants.

49 Detection and quantification of mRNA from environmental samples by qPCR has been reported to be three orders of magnitude more sensitive than hybridization techniques. But RT-qPCR has not yet been directly applied to environmental samples.

50 However, a few studies have used it to monitor genes in pure cultures: - carbon fixation in diatoms and pelagophytes by targeting rbcL, the gene encoding the large subunit of ribulose- 1,5-bisphosphate carboxylase/oxygenase (RubisCO). - dissimilatory (bi)sulphate reductase (DSR) gene expression was quantified during the growth of Desulfobacterium autotrophicum under a variety of different conditions.