629 CEE Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
QUANTITATIVE REAL TIME PCR
Tuesday, 11 October Room 3231 Engineering Hall (Environmental Engineering Teaching Lab)
Background
Standard PCR of the type that you set up in Lab 2 is not quantitative. That is, the number of copies present at the end of the reaction is not proportional to the original template concentration. It is not proportional because the reagents required to carry out the amplification (e.g. dNTPs, polymerase) either become depleted or inactive (and therefore limiting) after around 25 or 30 cycles (Figure 1). Since it would be valuable to be able to relate the amount of product produced to the amount of template in the original sample, a strategy has been developed to monitor PCR amplification in real time, allowing for quantitative PCR.
Figure 1. A plot of (A) copy number versus cycle number and (B) log of copy number versus cycle number. Notice that the open squares represent points after which the reaction begins to saturate and no longer produces an exact doubling of target during each cycle. The principle behind the type of quantitative PCR (qPCR) we will carry out in this lab is quite straightforward. A fluorescent dye (SYBR Green I) is added to the PCR master mix. SYBR Green I fluoresces only when it is bound to double stranded nucleic acids, and thus fluorescence increases as more product is generated. A special thermal cycler is used, with a laser and detector attached to it. During each extension step, the laser shines a specific wavelength of light into each tube, and the detector measures the resulting fluorescence. A computer records the fluorescence at each cycle, and special software plots fluorescence as a function of cycle number (in real time!). At the end of a PCR run, the software allows you to inspect the data. Data analysis Inspect Figure 1 more closely. Notice that the relationship between log(copy number) and cycle number is no longer linear after around 27 cycles (Figure 1B), even though it looks somewhat exponential until around 32 cycles, in Figure 1A. Thus, if we are visualizing raw copy number versus cycle number, we should focus only on the point when it just begins to rise above the
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
detection limit. This will vary based on the length of the amplicon (since fluorescence will be proportional to product length). In qPCR, the threshold is defined and adjusted to a value above the background and significantly below the plateau of the amplification curve (Figure 2A), thus it represents the detectable log-linear range of PCR. The threshold cycle (CT) is the cycle at which the amplification curve crosses the threshold. Therefore, higher target gene concentration in the template will have lower CT value because the amplification and the resulting fluorescent signal reach the threshold within less cycles for samples with higher target gene concentration, and thus exhibit a negatively proportional relationship (Figure 2 C). To quantify the target gene in a sample, generally, we must run a series of standards during each qPCR run. This is similar to the process of constructing a standard curve any time you are using a spectrophotometer (e.g. COD or soluble phosphate analysis). The standards consist of a positive control (usually a plasmid or PCR product containing the target we are trying to amplify from the sample) serially diluted by around 1:3 or 1:5. The positive control has been accurately quantified previously, and it is possible to calculate the number of copies in each PCR tube. An example of real time qPCR data is shown in Figure 2, on the next page. Quality control Correlation coefficient The correlation coefficient is a measure of how well the data from the experiment fit to the values assigned to the standards. The correlation coefficient should be a value between 0 and 1. The higher the correlation coefficient the more accurately the experimental data fit to expected values. Good correlation coefficient should be high than 0.990. Efficiency If PCR takes place at 100% efficiency, the template doubles after each cycle during exponential amplification. However, PCR efficiency can be influenced by factors such as the length of the amplicon, the GC content of the amplicon, secondary structure, non-specific amplification, PCR inhibitors in the template, activity of DNA polymerase, non-optimal reagent concentrations and cycle conditions. For example, PCR At 80% efficiency, the amount of DNA will increase from 1 to 1.8 after each cycle. Small difference in efficiency leads to big difference in the amount of final product. For example, each 10% lowering results in less than 25% of the previous column after 30 cycles. Therefore, for accurate quantification, PCR efficiency should be as close to 100% as possible. The PCR efficiency can be obtained from the slope of the standard curve we generated. The slope of the standard curve is related to the efficiency through the following equation:
(
Efficiency = 10
1 ) slope
1
A slope of -3.322 represents an efficiency of 100%. A higher absolute value of the slope (|s| ) will yield an efficiency of less than 100%. A lower |s| will yield an efficiency that is greater than 100%. In either case, the reaction should be optimized for better PCR efficiency.
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
Standards h Unknown k Negative control Template with 2 mismatches to primer set f Negative control Sterile dd water as template
(A) Fluorescence versus cycle number
Standards h Unknown k Negative control Template with 2 mismatches to primer set f Negative control Sterile dd water as template
(B) Log of fluorescence versus cycle number
(C) Threshold cycle (CT) versus target gene copy number (standard curve). Figure 2. An example of real-time PCR quantification
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
Melt Curve Melt curve is a tool to measure the melting temperature (Tm) of double stranded DNA molecules. When DNA duplexes incorporates of DNA-binding dyes (e.g. SYBR Green I), the fluorescence is brightest when the two strands of DNA are annealed. As the temperature is raised towards the Tm of the duplex, the DNA begins to denature and release SYBR Green I, causing a decline in fluorescence. At the Tm, there is a dramatic reduction in the fluorescence. Plotting the negative first derivative (-dF/dT) versus temperature will yield visible peaks, representing the Tm of the doublestranded DNA complexes. The Tm for each peak is dependent on the length of the amplified DNA as well as the GC content of the sequence. Primer dimers are shorter in length, thus melt at a much lower Tm than the intended product and are therefore easy to distinguish (e.g. the small peak at 80oC in Figure 3). With the melt curve, we can distinguish fluorescent signals of amplified products from primer dimers. The melt curve is usually generated after PCR amplification step is finished. The temperature of PCR reaction is increased every step by a small interval (e.g. 0.5oC) repetitively until reaching 95oC. The change of fluorescence signal is recorded by the detector in a real-time basis.
Figure 3 An example of melt curve (http://pathmicro.med.sc.edu/pcr/realtime-home.htm) Other ways to carry out qPCR Other types of qPCR have been developed, including TaqMan assays and molecular beacons. These methods are beyond the scope of this class, but may be of interest especially if more specificity is required since they often include detection using a specific fluorescent probe in addition to amplification with specific primers.
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
Lab format
There will be two stations set up in the teaching lab. You will be divided into two teams: A and B. Team member duties: Listens Leader: carefully to instructions given throughout the lab. Directs the other team members. Provides guidance (this person will have prior experience with PCR and/or gels) Records each step of the protocol as it is carried out. Makes copies of lab notes for other team members after class.
Scribe:
Experimentalist: Carries out most of the hands-on manipulations associated with the protocol. In this lab, we are going to quantify Accumulibacter phosphatis polyphosphate kinase (PPK) and bacterial SSU rRNA genes in activated sludge from a lab-scale enhanced biological phosphorus removal (EBPR) reactor from which we extracted DNA and conducted regular PCR in Lab 1 and 2 respectively. Remember that when we extracted DNA in Lab 1, the reactor was exhibiting very poor EBPR activity. A DNA sample collected when the reactor had high EBPR activity is also available and we will include it in our analysis to compare results from unhealthy and healthy EBPR sludges. Group A : Quantify bacterial SSU rRNA genes using primer 341-F and 534-R. This primer set will amplify bacterial SSU rDNA genes from each bacterial genome and give an amplified fragment with the length of 193 bp. Group B: Quantify Accumulibacter phosphatis PPK using primer ppk254-F and ppk1357-R. This primer set will amplify an internal fragment of the polyphosphate kinase gene (ppk) from A. phosphatis (the dominant organism in the bioreactor) genomic DNA. The full-length gene is around 2.1 kb in length, and the amplified fragment should be around 1.1 kb.
Protocol
1) Prepare the master mix. Assemble the master mix by sequentially adding the components as they are listed in Table 1. Invert the master mix tube several times to make sure it is mixed well. 2) Dispense the master mix. Aliquot 24 ul of mix into the assigned wells on a 96well thin-wall PCR plate according to Table 2. Using the right positions on the plate is important because you need to tell the real-time PCR machine which wells the detector should record signals from.
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
3) Add the template. Add 1 ul of template into the well according Table 2. When adding the template, make sure that your pipette tip is under the liquid surface in the well. Mix the template with PCR mix by pipetting up and down for 7 times. Notice that we duplicate both standards and samples because it is observed that the plate is not uniform and small differences may cause well to well variation. We need to consider the non-uniformity by duplicating. 4) Place a sheet of optical sealing tape on the top of the 96-well plate. Use the tape applicator (flat plastic wedge) to smooth the tape surface and tightly seal the tape on the plate. Avoid touching the surface of the sealing tape with gloved fingers. Tear off the white strips that remain on the sides of the tape. Now it is ready to put the plate into the real-time PCR machine (Bio-Rad iCycler).
Table 1 Master Mix
Component (concentration) pure sterile dd water 10X PCR buffer (no MgCl2) 25 mM MgCl2 (25 mM) 10 mM dNTPs (10 mM each) SYBR Green I (10X) Fluorescein (1 uM) DMSO
2 1
Final Conc
Group A: 16S rDNA 25 l 30 reactions reaction (L) (L) 12.5 375 75 90 60 30 7.5 0 37.5 37.5 7.5 2.5 3 2 1 0.25 0 1.25 1.25 0.25 1 25 L
Group B: ppk 25 l 32 reactions reaction (L) (L) 11.25 2.5 3 2 1 0.25 1.25 1.25 1.25 0.25 1 25 L 360 80 96 64 32 8 40 40 40 8
1X 3 mM 0.2 mM 0.4X 10 nM 0 or 5% 500 nM 500 nM
Forward primer (10 M) Reverse primer (10 M)
AmpliTaq Gold DNA polymerase
Template Total
1
Fluorescein is added for well-factor collection on the real-time PCR detection systems. Well factors are used to compensate for any system or pipetting non-uniformity in order to optimize fluorescent data quality and analysis.
2
DMSO (Dimethylsulfoxide) is added to PPK amplification because PPK gene has a high GC content and DMSO can reduce both the melting temperature and any inhibitory secondary structure.
CEE 629 Fall 2005 Environmental Microbial Biotechnology
Lab 3 Quantitative Real Time PCR
Table 2 Wells to add PCR mix and template
Group A:
16S rDNA Standards Samples in Table 3 16S rDNA Standards Samples in Table 3 A1, 108 copies B1, #1 E1, 108 copies F1, #1 A2, 107 copies B2, #2 E2, 107 copies F2, #2 A3, 106 copies B3, #3 E3, 106 copies F3, #3 A4, 105 copies B4, #4 E4, 105 copies F4, #4 A5, 104 copies B5, #5 E5, 104 copies F5, #5 A6, 103 copies B6, #6 E6, 103 copies F6, #6 B7, #7
F7, #7
Group B:
PPK gene Standards Samples in Table 2 PPK gene Standards Samples in Table 3 A1, 10 copies B1, #1
7
A2, 10 copies B2, #2
6
A3, 10 copies B3, #3
5
A4, 10 copies B4, #4
4
A5, 10 copies B5, #5
3
A6, 10 copies B6, #6
2
B7, #7
B8, #8
E1, 107 copies F1, #1
E2, 106 copies F2, #2
E3, 105 copies F3, #3
E4, 104...