Objectives Slide notes: Page 1 of 41
Module 1: The Basics Of Real Time PCR Slide notes: Module 1: The Basics of real time PCR Page 2 of 41
Polymerase Chain Reaction Slide notes: Here is a review of PCR, The classic method of amplifying DNA involves 3 steps:. Strand denaturation, Primer annealing, and Primer extension. This is typically performed for 30 to 40 cycles. PCR generates exponential amplification of product in each cycle, which creates a doubling of PCR product until depletion of PCR reagent components. Page 3 of 41
Polymerase Chain Reaction Slide notes: Lets look at what happens during a PCR reaction. Initially the reaction reagents are very fresh and product formation becomes exponential. Eventually a reagent becomes limiting and products are no longer doubling. This is called the Linear phase. The reaction eventually stops and this is called the Plateau phase. Typically, end-point or gel PCR measurements are taken during the linear phase. Page 4 of 41
Time Point of PCR Analysis Slide notes: However, the most accurate place to quantify PCR would be in the exponential phase where there is an exact doubling of product. The reaction is proceeding at a rapid rate and is very precise. Measurements in the plateau phase indicate the end of the PCR and the signal could be variable as we see with these 96 replicates. Page 5 of 41
Signal Generation with a TaqMan Probe Slide notes: The way Applied Biosystems accomplishes the measurement of the exponential phase is by using the 5 nuclease assay. The 5 nuclease assay requires two things to function, FRET and 5 nuclease Activity of AmpliTaq Gold Polymerase. Page 6 of 41
1. Fluorescent Resonance Energy Transfer (FRET) Slide notes: Lets look at FRET. The premise is that when dyes are excited, energy from a high energy dye is transferred to that of a low energy dye when they are in close proximity. When the dyes are separated, the energy transfer stops and the high energy dye fluoresces brightly once again. Text Captions: Energy Transfer from High to Low High Energy dye Energy Transfer Reverses Excitation Low Energy Dye Dyes Separate Excitation Page 7 of 41
2. 5 Nuclease Activity Slide notes: The 5 nuclease activity of TAQ is the second feature that makes the 5 nuclease assay work. When the TAQ extends off of a primer, when it encounters a molecule that is hydrogen bound to the DNA, the nuclease activity is triggered and the enzyme chews apart the molecule blocking extension. 5 nuclease activity does not harm PCR products. The activity is only triggered when the TAQ molecule is extending from a 3 hyrdoxyl group on the end of a primer, and when the TAQ encounters a hydrogen bound molecule in it s path. Text Captions: Polymerase Nucleic Acid Primer Hydrogen bound nucleic acid 3' Hydroxyl Group 5' Hydrogen bond Page 8 of 41
5 Nuclease Assay Using TaqMan probes Slide notes: The 5 nuclease assay takes advantage of FRET and 5 nuclease activity by using a fluorescent labeled probe. The probe is an oligo roughly 13-25 bases that binds on the template between the Forward and Reverse primers.the Probe gives an added specificity level to the assay. Text Captions: Oligo Page 9 of 41
5 Nuclease Assay Using TaqMan probes Slide notes: The probe is labeled with a High energy reporter dye on the 5 end and a Low energy quencher dye on the 3. When the probe is intact, the FRET technology keeps the reporter from shining too brightly by transferring its energy to the quencher. The quencher will emit this energy, either as light or heat. In the intact probe, the reporter dye will emit only a very low level of fluorescence. Text Captions: The Reporter dye is a high energy dye. The Quencher dye is a low energy dye Energy of the Reporter dye is transferred to the Quencher dye Energy is emitted as light or heat Reporter energy is quenched. Intact Probe Intact Probe Page 10 of 41
5 Nuclease Assay using TaqMan probes Slide notes: During PCR the Taq extends from the forward primer, bumps into the probe making a fork like structure, exposing the hydrogen bond, and then begins to cleave the probe Text Captions: Extension Fork-like structure Page 11 of 41
5 Nuclease Assay using TaqMan probes Slide notes: Once the probe is cleaved, the Reporter dye is separated from the quencher. The Reporter dye then emits its fluorescent energy at full strength. Text Captions: Probe is being cleaved by the polymerase Reporter is able to emit energy Page 12 of 41
5 Nuclease Assay using TaqMan probes Slide notes: Text Captions: One probe is cleaved for each copy of DNA polymerized. Page 13 of 41
Fluorescence Changes due to FRET Slide notes: This slide demonstrates the fluorescent changes that are given off when the probe is intact and cleaved. Initially in an intact probe you see a lowered reporter fluorescence and a higher quencher signal. When the probe is cleaved the reporter increases in signal and the quencher decreases in signal. Text Captions: Reporter fluorescence is lower than Quencher fluorescence Reporter fluorescence is higher than Quencher fluorescence Page 14 of 41
Increase in Reporter Signal Reports Amplification of Target Slide notes: This fluorescence is captured by the instrument and a signal is generated above background levels. An increase in signal over time is displayed on the instrument as the instrument captures the increase in reporter signal. Text Captions: Multicomponent view of instrument software Increase in reporter fluorescence signal Page 15 of 41
Slide 16 Slide notes: Another technology used to perform real-time PCR is the SYBR Green I dye Assay. SYBR Green dye binds to double stranded DNA and fluoresces only when the DNA is in a double stranded state. It binds to the minor groove of the DNA molecule. Because of this characteristic, SYBR Green Dye will bind to all double-stranded DNA and is not specific to targeted amplicons. Page 16 of 41
Slide 17 Slide notes: During PCR, when the DNA is denatured SYBR Green dye falls off of the DNA and does not fluoresce. During the annealing step, when the DNA re-anneals SYBR Green dye will bind and fluoresce. This makes the fluorescent signal transient. The specific product being amplified by the primers increases throughout the pcr, the PCR product is double stranded and SYBR Green dye will bind the product. The product increases over time and SYBR Green dye signal will be seen as an amplification above background. Page 17 of 41
Increase in SYBR Green Dye Fluorescence Reports Amplification of Target Slide notes: As Amplicons accumulates, more SYBR Green Dye signal is captured and displayed as seen here on the instrument. Text Captions: Multicomponent view of instrument software Increase in signal Page 18 of 41
Specificity check SYBR Green 1 dye Slide notes: SYBR Green assays are inherently non- specific. In other words, any double-stranded DNA would generate a signal. Therefore, a melting curve or gel analysis should be done to determine whether or not there is primer dimer formation or a secondary product. A peak in a single location would indicate one product as would one band on a gel. Text Captions: One peak location for all samples One band size for all samples Page 19 of 41
Chemistry Comparison Slide notes: So, which chemistry should you choose? TaqMan assays provides extra specificity because of the probe and is ideal for SNP detection, multiplexing with two different targets and plus minus assays. Optimization is minimal as the universal guidelines can be used during assay development. Because Sybr green dye assays rely only on primers for specificity, extra steps should be taken to ensure specificity. Sybr Green dye also uses universal development guidelines, but includes checking for primer dimer formation and using less primer. Page 20 of 41
From Fluorescence to Results: Terminology Slide notes: In viewing an amplification plot, there are several features with which you need to become familiar. On the graph, the Y axis shows the Normalized reporter signal and the X axis shows the number of PCR cycles. The Baseline is a region of the PCR cycles (default 3-15) where the instrument takes a measurement of background fluorescence resulting from the block, consumables, or reagents. This baseline is used to normalize the initial fluorescence of the reaction. It enables the analysis to account for background fluorescence while detecting the actual PCR reaction above background. Rn is the reporter signal normalized to the passive reference dye, ROX Dye, from our master mix. The drn is the normalized reporter signal with background subtracted. Next is the Threshold. The threshold is a level of fluorescence where the PCR is measured in the exponential phase. The threshold is movable and default value is.2. The Ct value is the PCR cycle where the fluorescence from the amplification crosses the threshold. Text Captions: Amplification is crossing threshold Page 21 of 41
From Fluorescence to Results Step 1 Determination of Ct Value Slide notes: Step 1 of an analysis would be to determine the CT value of a sample. The software will assign this to a sample based on where the sample crosses the threshold. For instance this sample crosses the threshold at cycle 27.5 so its Ct value would be 27.5. Text Captions: Sample crosses threshold at 27.5 cycles Page 22 of 41
From Fluorescence to Results Step 2 Comparison of Ct Values Slide notes: Step 2 is the comparison of Ct values between samples. If you are performing quantitative PCR, Ct values would be obtained for each standard. This case shows 10 fold dilutions of a known amount. Ct values are assigned for each point in the dilution. The standards are then plotted in a regression line plot. An unknown would be run on the same plate with the standards and a Ct value would be assigned for that unknown. The Ct value of the unknown is then compared to the standard curve regression line and a quantity can then be assigned to the unknown. Text Captions: 10-fold dilutions of a standard Regression line of standard curve Unknown sample Unknown Ct value is converted to a quantity Page 23 of 41
Efficiency Matters: Theory of Quantitative PCR Slide notes: Next we will talk a little about efficiency of PCR. PCR efficiency affects the quantitative outcomes of a PCR assay. In the equation above: y =(x(1+e)n is the equation for efficiency. We are interested relating the quantity or Yield (y) to the amount of starting material (x) for each sample. The rest of the variables need to be known to quantify the yield. n is the number of cycles it takes for a sample to cross the threshold, the Ct value. e is the efficiency. Efficiency is very important and should be constant or close to 100%. Efficiency can be optimized by using the AB Rapid Assay Development Guidelines. If the efficiency is 100%, then efficiency will be a constant among the reactions and quantitation of x is possible. Page 24 of 41
Effect of Amplification Efficiency on Results Slide notes: Now lets look at how efficiency effects PCR. If you had an input amount of 100 copies (x) and the reaction was 90% efficient (e) and your Ct value was 30 (n) you would get 23 billion copies. If the same reaction were only 80% efficient you would get 4.6 billion copies. This results in a difference of 5-fold!! So you can see from a 10% efficiency difference, comes a dramatic change in results. Efficiency really affects quantitation of samples. Page 25 of 41
Relationship between Initial Copy Number and Cycle Number Slide notes: To help understand why efficiency is important, it is first important to under stand that if a PCR reaction is operating at 100% efficiency, there will be a doubling of product for each cycle of PCR. In the slide, if 1000 copies came up at cycle 28, then it would take one more doubling of product, (one more cycle) for the 500 copy sample to be visible or catch up to the 1000 copy sample. The same would happen for the 250 copy sample. There should be a 1 cycle difference between each sample with a 2 fold dilution. Page 26 of 41
Amplification Efficiency Effects Slide notes: Now look at two examples of efficiency issues. Theoretically in 100% efficient PCR reaction, 1 cycle = a 2 fold change, a 10 fold dilution series should then take about 3.3 cycles between dilutions. If the efficiency were affected and reduced to 80% it would take longer cycle times between the two dilutions, or about 3.9 cycles. Page 27 of 41
Rapid Assay Development Guidelines Slide notes: To account for these efficiency issues, AB has taken out much of the guess work. We have developed software and chemistries to help with optimization to achieve close to 100% efficiency of pcr reactions. These include Universal Master Mixes, universal Assay Conditions, Probe and Primer design parameters, and Universal Thermal cycling Conditions. Page 28 of 41
Slide 1 Slide notes: The Universal master mix contains all PCR reagents except primers, probe and sample, and is fully optimized to work with Primers and Probes designed by Primer Express Software, Genomic Assays and Universal thermal cycling parameters. The master mix contains AmpliTaq Gold Polymerase and the ROX Passive reference Dye 1.Note that you would use SYBR Green master mix if you chose SYBR Green Dye Page 29 of 41
Passive Reference ROX Dye Slide notes: The function of ROX dye is to improve precision by accounting for small fluorescent changes. Rox does not participate in the PCR reaction. For instance in Replicate 1, the FAM dye signal seems higher than Replicate 2 and without correct would imply different results. But if we use ROX dye we see that the ROX dye also changed along with the FAM dye signal. This indicates a possible pipetting error or other issues, like bubbles in the well or optical differences. We then divide the Reporter signal by the ROX dye signal to normalize the replicates. Page 30 of 41
Why Use a Passive Reference? Slide notes: After normalization, we see now that the replicates agree and precision is good. The yield is now the same between the 2 replicates. Text Captions: Replicate fluorescence levels now are the same. Page 31 of 41
With ROXTM dye Slide notes: Here is an example of a standard curve using ROX dye normalization and one without the normalization. Note that the precision is affected in the samples without ROX dye. In the samples normalized to ROX dye there is clear separation of each standard. Text Captions: Standard Curve normalized to ROX dye Standard Curve not normalized to ROX dye Clear separation and precision of dilutions Dilutions are not clearly defined Page 32 of 41
The Benefit of ROXTM dye: Increased Precision Slide notes: Here is another example of replicates from the 5000 copy standard in our RNase P plate. RNase P is a 2 copy human gene. Note that without ROX dye the replicates seem tight, but have a relatively high Standard deviation. With ROX dye normalization, the replicates are now more precise, within 0.3 CT of each other and the Std dev has improved.. Text Captions: Replicates not very precise. Replicates have good precision Replicates Ct values are within 0.3 Page 33 of 41
Universal Primer and Probe Concentrations: Universal Assay Conditions Slide notes: We also utilize universal Primer and probe concentrations. These concentrations mean little or no optimization required during assay development. This means your time to results will decrease. Probe Concentrations are 250nM probe and 900nM each primer. Page 34 of 41
Slide 35 Slide notes: These are our oligo design parameters: AB experimented with design and determined that these guidelines will help to achieve 100% efficiency with minimal optimization. All primers are between 58-60. No more than 2 G's or C's at the 3 end of the primer. Amplicon length recommended 150 base pairs or less. Short amplicons create higher efficiency assays. Probes are 10C higher than the primer T. No G on the 5 end, Select a probe with more C s than G s. Reporter is on the 5 end and Quencher on the 3 end. For SYBR green assays you would design the primers using primer express and the same guidelines apply. SYBR Green assays may need to use less primer to reduce primer dimer and non-specific binding. Primer Express software incorporates all these parameters. AB also has a Custom design program called File Builder or readily available Genomic assays that can be ordered on line. Text Captions: To design a SYBR Green Assay, use primers generated from Primer Express. Assay may require less primer in the reaction. See the Primer Express Software User Guide for more information. Page 35 of 41
Copy of Assays Slide notes: Page 36 of 41
Probe Design Guidelines Slide notes: Reporter dye choices are FAM, VIC and NED. Quencher choices are TAMRA dye and the alternate quencher choice of MGB-NFQ which contains a non-fluorescent quencher. Text Captions: These are Applied Biosystems available reporter dye choices. Page 37 of 41
Slide 38 Slide notes: MBG-NFQ probes contain a molecule that binds to the minor groove of the amplicon-probe moiety. This changes the effective melting temperature of the probe by 10 to 15 degrees allowing smaller probe design with the same Tm as the longer probes.the MGB wraps around the last six bases of the probe to create the moiety that effects Tm. The MGB is attached to a non-fluorescent quencher that emits heat instead of light. Because of this technology, MGB-NFQ probes are more specific, and especially valuable when performing SNP assays where the discrimination is usually a single base. Page 38 of 41
Universal Thermal Cycling Protocol Slide notes: We also use universal Thermal Cycling conditions. 95 degrees for 10 minutes activates the AmpliTaq Gold Polymerase. The PCR is performed at 95C for 15 sec and an anneal/extend step at 60C for 1 minute. The AmpliTaq Gold Polymerase easily extends 150 bases at a temp of 60C. We perform the anneal/extend step for one minute to ensure all the possible extensions are completed, which means all the possible probes have been cleaved. This allows for accurate quantification. Page 39 of 41
Trademarks Slide notes: Page 40 of 41
Slide 41 Slide notes: Text Captions: Congratulations for finishing Module 1! Please continue to Module 2: Real-Time Quantification. Page 41 of 41