LIGHTCYCLER EXPERIMENTAL

Transcription

1 LIGHTCYCLER EXPERIMENTAL D E S I G N CONTENTS PART 1 INTRODUCTION Introduction to fluorescence applications for the LightCycler Fluorescence techniques for the LightCycler Double strand DNA dyes Sequence specific probes Selection of fluorophores 8 PART 2 FLUORESCENCE APPLICATIONS FOR THE LIGHTCYCLER Principles of product identification and purity determination Using SYBR Green I for product ID and purity determination 9 Using product melting temperature as a measure of purity 10 Using SYBR Green I and melting curves to optimize PCR 12 Estimation of product melting temperature.12 Presenting melting curve dataas melting peaks.13 Using melting peaks to optimize PCR reactions.14 Multiplexing by melting temperature with SYBR Green I..14 Two important things to control for in SYBR Green I melting curve experiments Using sequence specific probes for product identification.17 Designing hybridization probes for product identification.18 Monitoring fluorescence 19 Experimental Design - 1

2 2.2 Principles of mutation detection using hybridization probes Using FRET hybridization probes for mutation detection...20 Designing hybridization probes for mutation detection Principles of quantification using fluorescence Setting up a quantification experiment..23 Selecting standards 23 Determining copy number from fluorescence data Selecting a fluorescence method for quantification 24 Quantification with SYBR Green I 24 Increasing specificity with SYBR Green I...27 Quantification with sequence specific probes 27 Designing hybridization probes for quantification.28 Monitoring fluorescence 28 PART 3 FLUORESCENCE MONITORING SYSTEMS Using SYBR Green I with the LightCycler..29 Recommended concentrations of SYBR Green I..31 Influence of Magnesium ion concentration on SYBR Green I fluorescence Using hybridization probes with the LightCycler Design and synthesis of fluorescently labeled oligonucleotides...33 Design of adjacent hybridization probes..33 Synthesis of adjacent hybridization probes 35 Hybridization probes designed as a primer-probe system 36 Designing fluorescently labeled primers 37 Designing single hybridization probes 37 Experimental Design - 2

3 EXPERIMENTAL DESIGN FOR THE LIGHTCYCLER This chapter provides a broad introduction to fluorescence applications in PCR that are ideally suited for use with the LightCycler. Several applications coupling PCR amplification and fluorescence monitoring are presented, including product purity determination, single base pair mutation detection and PCR quantification. Part 1 of this chapter describes the fluorescence techniques that are recommended for use with the LightCycler including the dsdna dye SYBR Green I, and fluorescently-labeled sequence-specific probes. Part 2 of this chapter contains Experimental Design suggestions for three common LightCycler applications Product identification and purity determination Mutation detection using hybridization probes PCR quantification Part 3 of this chapter provides detailed information on fluorescent probes used in the LightCycler, including recommended conditions for SYBR Green I (Part 3.1), and hybridization probes, including probe design and synthesis (Part 3.2). Experimental Design - 3

4 PART 1 INTRODUCTION 1.1 Introduction to Fluorescence Applications for the LightCycler The LightCycler couples rapid PCR amplification with fluorescence monitoring of PCR reactions in real-time. Two fluorescence techniques are widely applicable: Dyes that fluoresce upon binding to double-strand DNA Sequence-specific fluorescent probes that recognize amplified DNA sequences. Both fluorescence techniques can be used with the LightCycler. There are several advantages to monitoring PCR using fluorescence, the most immediate is the ability to confirm amplification of target DNA as it occurs without the need for any post- PCR analytical techniques such as gel electrophoresis. This can be done using either dsdna dyes or fluorescent probes (see Part 2.1 of this chapter). The LightCycler can also be used for rapid genotyping. Products differing by a single base can be differentiated using fluorescently labeled hybridization (see Part 2.2 of this chapter). The fluorescent signal generated during PCR amplification can also be used for product quantification. Quantification of starting template concentrations down to the single copy level is possible with the LightCycler using either dsdna dyes or sequencespecific hybridization probes (see Part 2.3 of this chapter). Experimental Design - 4

5 The LightCycler can be used for rapid genotyping. Products differing by a single base can be differentiated using fluorescently labeled hybridization probes. Recommendations for experimental design for all three of the applications described above are described in detail in Part 2 of this chapter. 1.2 Fluorescence Techniques for the LightCycler Double strand DNA dyes. Several dyes are available that generate a fluorescent signal on binding to double strand DNA (Figure1a). These dyes have the advantage of recognizing any dsdna product and are also extremely economical to use. Only small amounts of dye need to be included in a reaction to produce a fluorescent signal. The lack of specificity of dsdna dyes can present some problems, however. Unless a PCR reaction is highly optimized, increases in fluorescence during amplification may not always reflect the formation of a specific DNA product. This disadvantage can be overcome on the LightCycler by monitoring the melting temperature of reaction products formed. Since melting temperature (Tm) is a good indicator of product GC percentage and to a minor extent product length and sequence, specific products can be identified on the basis of Tm. The ability to monitor amplification on the LightCycler with dsdna dyes provides investigators with a convenient and inexpensive way to follow PCR without having to synthesize expensive, fluorescently labeled probes. The double strand DNA dye SYBR Green I (Molecular Probes, Eugene, OR) is recommended for use with the LightCycler. SYBR Green I can be used for product identification, product purity determination and PCR quantification on the LightCycler. Experimental Design - 5

6 Figure 1 Before After A dsdna Dyes S S B Hairpin Primer D Q D Q C Hairpin Probe D Q D Q D Hydrolysis D Q D Q E Hybridization A D D A Sequence specific probes. PCR amplification can also be monitored on the LightCycler using sequence-specific fluorescent probes. Probes have the advantage of complete sequence specificity but may be difficult and expensive to synthesize. Several probe designs are available to users: 1. Hybridization probes. The hybridization probe system was specifically designed for the LightCycler. Two fluorescently labeled oligonucleotides recognize adjacent sequences within an amplicon internal to the PCR primers (Figure 1 E). One probe is labeled at the 3 end and the other at the 5 end. The signal appears during hybridization by resonance energy transfer when the fluorophores are brought into close proximity. Hybridization probes have been used successfully with the LightCycler for product identification and quantification. Since the fluorescent signal is directly related to product concentration, these probes are very useful for low copy PCR quantification. Hybridization probes have also been used with the LightCycler for rapid genotyping and mutation detection. When hybridized to a template, fluorescence resonance energy transfer between labeled, sequence-specific probes generates a fluorescent signal. If the temperature is raised slowly, with continuous fluorescence monitoring, it is pos- Experimental Design - 6

7 sible to follow loss of fluorescence as probe/product hybrids melt apart. The melting temperature (Tm) of a probe can be determined to within a fraction of a degree using this technique. A single base mismatch between probe and template results in a probe Tm shift of several degrees. One modification of the hybridization probe design is to use one labeled primer and one labeled probe, instead of two labeled probes. In this design the labeled probe hybridizes to the labeled amplicon strand formed from extending the labeled primer. Resonance energy transfer occurs between fluorophores attached to opposite strands of DNA, rather than between fluorophores attached to oligonucleotides hybridized to the same strand. 2. The fluorescence resonance energy transfer of hybridization probes arises from transfer of energy from a donor to an accepter fluorophore. In contrast, the probe systems described below are based on the interaction between a fluorophore and a quencher. A fluorescent signal is generated when two dyes are separated either by distance or by exonuclease hydrolysis. Although the LightCycler is not optimized for these probes and they are more difficult to synthesize, these alternative probes can be used in the LightCycler. For more information on any of these labeled oligonucleotide probes, refer to the respective manufacturer s instructions. 3. Exonuclease probes (TaqMan, Roche). These probes are designed with a fluorophore (fluorescein) and a quencher (rhodamine) on the same sequence-specific probe (Figure 1 D). During the annealing/extension phase of the PCR reaction, the 5 to 3 exonuclease activity of Taq polymerase cleaves the probe between the dyes releasing the fluorescein from quenching. The fluorescent signal increases as amplification proceeds and is related to product formation. 4. Hairpin probes (Molecular Beacons ). These probes are labeled at either end with a fluorophore and a quencher, and are similar in design to the primers described above (Figure 1 C). The probes recognize sequences within the amplicon. When they hybridize to the target sequence during amplification, the fluorophore is separated from the quencher and a fluorescent signal is generated. Experimental Design - 7

8 5. Other probes (not sequence-specific, just PCR primer-specific). Hairpin Primers (Sunrise Primers, Oncor). These are hairpin primers labeled with a fluorophore at one end and an opposing quencher within the primer sequence (Figure 1 B). Specificity depends on the specificity of the primers. A fluorescent signal is obtained during amplification when the hairpin is linearized and the fluorophore and the quencher are separated by distance. An increase in fluorescent signal correlates directly with the formation of product. Selection of Fluorophores The LightCycler has three channels available for fluorescence detection. Channel 1 (F1) detects emissions from fluorescein or SYBR Green I (Molecular Probes) and Channel 2 (F2) detects emission from Cy5 (Amersham). Dyes for use with Channel 3 (F3) will be available soon. For more information on labeling oligonucleotides with fluorescent dyes appropriate for the LightCycler please see Part 3 of this chapter. Probe Summary Table Hybridization Probes SYBR Green I Two-Probe Primer-Probe Exonuclease Probe Hairpin Probe Hairpin Primer Mechanism ds DNA Binding FRET FRET Quenching Quenching Quenching Probe Design Probe-free Two probes with single labels One singlylabeled primer and one singlylabeled probe One duallabeled probe One duallabeled probe One duallabeled probe Relationship to Product Concentration direct Direct 3rd order Direct 2nd order Cumulative Cumulative Proportional Sequence Specific No Yes Yes Yes Yes No Synthesis Difficulty None Easy Moderate Difficult Difficult Difficult Cost Low Moderate Moderate High High High Experimental Design - 8

9 PART 2 FLUORESCENCE APPLICATIONS FOR THE LIGHTCYCLER 2.1 Principles of product identification and purity determination Both dsdna dyes and sequence specific fluorescent probes can be used for product identification. In the case of dsdna dyes the melting temperature of the product is used to confirm identity. When fluorescent probes are used, product formation is confirmed by following the generation of a fluorescent signal during amplification. The relevant strengths and weaknesses of these two fluorescence methods are described below to help you decide which method best suits your application Using SYBR Green I for product identification and purity determination Advantages of SYBR Green I Inexpensive Easy to use. Simply include diluted dye in the reaction as an additional reagent. Reactions can be followed on the LightCycler without the synthesis of any fluorescently labeled probes. Ideal for optimizing amplification reactions. Disadvantages of SYBR Green I No sequence specificity. Experimental Design - 9

10 Double strand DNA (dsdna) products can be monitored on the LightCycler using a dsdna dye such as SYBR Green I. This dye is especially attractive due to its lack of sequence selectivity and its compatibility with PCR. By including SYBR Green I in your PCR reaction, product purity can be confirmed simply by monitoring the melting temperature (T m ) of your product after amplification, often eliminating the need for any gel-based post-pcr analysis to confirm product purity. Product melting temperature is determined by monitoring fluorescence with increasing temperature, a sharp loss of fluorescence is observed as the double strand product melts apart. SYBR Green I provides an economical method for monitoring PCR products since it does not require the synthesis of any fluorescently-labeled probes. One useful feature of SYBR Green I is that it recognizes all dsdna molecules including primer-dimers and other nonspecific products. This dye can, therefore, be used to optimize PCR reactions by determining the conditions that minimize the formation of nonspecific products as determined by melting curve analysis. Using product melting temperature as a measure of purity DNA duplexes will melt apart into single strands at high temperatures, so unpairing of dsdna can be measured on the LightCycler by monitoring loss of SYBR Green I fluorescence with increasing temperature. The temperature at which a dsdna product melts apart is dependent on its GC content and length, so two products differing in GC content and length can often be differentiated by their melting temperature. Tm 0% GC A 41 o C d 100% GC 50 mer A 10 o C d 1000 mer The percent GC effect is greater than the length effect. Products with the same GC content that are close to the same length may not be distinguishable, especially for longer PCR products. Continuous monitoring of fluorescence as the temperature of the PCR product is slowly raised from annealing to denaturation allows the generation of a melting curve that can be used to determine the melting temperature of a product and confirm the specificity of the amplification. Experimental Design - 10

11 Product melting on the LightCycler is typically performed by programming a brief (0 second) denaturation step, followed by a short (5-30 second) hold at least 10 o C below the melting temperature of the product. The temperature is then raised slowly (0.2 degrees per second) to about 5 o C above the Tm of the product while fluorescence data is acquired. For details on programming a melting curve see the Programming section of this manual. Figure 2A illustrates SYBR Green I melting curves acquired for two amplicons, hepatitis B virus and b-globin and a mixture of the two amplicons. The two products vary in length and GC content as reflected in their different melting temperatures. Figure 2A A. Fluorescence HBV b-globin Mixed Product Temperature ( C) Experimental Design - 11

12 Figure 2B B Specific Product Fluorescence 6 4 Primer Dimer Temperature ( C) Using SYBR Green I and melting curves to optimize PCR reactions You can use SYBR Green I to optimize your PCR reaction. Figure 2B shows different melting curves obtained for specific versus nonspecific products. Nonspecific products such as primer dimers can be distinguished from larger specific products by the shape and positions of their respective melting curves. Mixtures of small, nonspecific products usually melt at lower temperatures and over a broad temperature range whereas homogeneous, specific PCR products show a sharp melting transition at higher temperatures. Estimation of product melting temperature When designing experiments where a melting curve is generated, it is useful to have an idea of the approximate T m of your desired PCR product. The melting temperature of a long (>100 base pairs) sequence of DNA can be predicted mathematically and several primer selection programs will estimate the T m of PCR products. Alternatively, use the equation described below to approximate your product T m. This equation is only accurate to a few degrees centigrade; the exact T m of your product can be determined empirically using the LightCycler. Tm = A (%GC) - (500 / bp)* * JG Wetmur, Formation and Structure of Nucleic acid Hybrids in Molecular Biology and Biotechnology, A Comprehensive Desk Reference. (RA Meyer, ed). VCH Publishers, New York, Experimental Design - 12

13 - Where: A = 16.6 x log10{[salt] / ( [Salt]} [Salt] = 4[Mg ++ - dntp] [Na + + K + + Tris + ] bp = length of product All concentrations are in Molar amounts. For Idaho Technology Buffers: 2 mm Mg ++ [Salt] = mm Mg ++ [Salt] = mm Mg ++ [Salt] = mm Mg ++ [Salt] = 0.28 Figure 3A. Three amplicons HBV b-globin df/dt 1.0 Mixed Product df/dt Temperature ( C) Differentiation of melting curve data allows it to be visuallized as melting peaks. It is often easier to determine the Tm of a product if melting curve data is presented as the negative value of df/dt. This is illustrated in Figure 3A, where melting curve data from Figure 2A are now presented as melting peaks. The peak value is the estimated Tm of a given product. If you open your data file using the LightCycler Melting Curves analysis software, a melting peak will automatically be generated for every sample you select. The LightCycler software allows you to determine the area under each curve. The area under the curve is related to product concentration. This type of analysis can be used to estimate relative concentrations of specific versus nonspecific products and for product quantification. Experimental Design - 13

14 Using melting peaks to optimize PCR reactions Melting peaks are extremely useful for optimizing PCR conditions. A poorly optimized reaction often results in the generation of large amounts of primer-dimers and nonspecific products at the expense of the specific product. These nonspecific products are easily distinguished by melting peak analysis because they tend to melt over a wide temperature range generating a broad melting peak at lower temperatures (see Figure 3B). Varying reaction conditions such as the magnesium concentration or the annealing temperature can improve the specificity of a PCR reaction. Including a melt after amplification can confirm the specificity of the reaction. In a perfectly optimized reaction only one peak (representing a specific product) should be observed at the predicted Tm. Figure 3B. Primer dimers and ns products Primer Dimer Specific Product df/dt Temperature ( C) Multiplexing by melting temperature with SYBR Green I In cases where an internal control is needed during amplification, two or more gene fragments can be amplified simultaneously (multiplexing). The amplification of multiple products can be followed on the LightCycler using SYBR Green I if the products differ sufficiently in melting temperature. The presence of several primer sets in the reaction mix can sometimes Experimental Design - 14

15 be problematic, leading to the formation of primer dimers and unwanted nonspecific products. Since a traditional hot start is difficult with LightCycler reactions, due to sample preparation conditions and the reaction cuvettes themselves, it is recommended that a reagents such as anti-taq antibodies or AmpliTaq Gold be included in the reaction mix to minimize the formation of nonspecific products. Figure 4 illustrates the simultaneous amplification of a cystic fibrosis product and a c-erb 2 amplicon using SYBR Green I. Notice the clear differentiation of the two products using melting peak analysis (for a detailed description of this experiment see Section 3B of the Fluorescence Protocols section of this manual). Figure 4 Fluorescence No Template CF c-erb CF c-erb df/dt No Template Temperature ( C) Experimental Design - 15

16 Two important things to control for in SYBR Green I melting curve experiments 1. SYBR Green I concentration. The concentration of SYBR Green I used in a reaction can influence the profile of a melting curve. Lower concentrations of dye (1: 30,000 dilutions of the Molecular Probes stock solution) are recommended as they produce more accurate and more reproducible melting curves. (Figure 5A). 2. Temperature ramp rates As noted above, subjecting a dsdna product to a steady increase in temperature generates a melting curve. Slow ramp rates (the rate at which the temperature is increased) between 0.1 C /sec and 0.5 C /sec are optimum, faster ramp rates increase the apparent Tm of a product (Figure 5B). If you have selected SYBR Green I for your application see Part 3.1 of this chapter for more details on how to use the dye. Examples of experiments that include product identification are described in detail in the Fluorescence Protocols section of this manual. Figure 5A :5,000 Fluorescence :40,000 1:10, :20, Temperature ( C) Experimental Design - 16

17 Figure 5B C/sec Fluorescence C/sec 2.0 C/sec 0.2 C/sec 0.5 C/sec Temperature ( C) 2.12 Using sequence-specific probes for product identification Advantages of oligonucleotide probes Sequence specificity. Sensitivity. Disadvantages of oligonucleotide probes Requires probe synthesis for each target Monitoring amplification with sequence-specific probes Fluorescently labeled oligonucleotide probes are recommended when sequence-specificity is a requirement. Labeled probes can be powerful tools for product identification. Keep in mind however that the sequence-specific nature of these probes does not allow detection of nonspecific products. They are only able to follow the formation of the PCR product to which they hybridize. This means that they can be used for product identification but have limited use in product purity determination. Experimental Design - 17

18 In order to follow hybridization of probes to PCR templates on the LightCycler, the probe or probes used must be fluorescently labeled. Several fluorescent probe designs have been described in the literature. Hybridization probes were specifically designed for use on the LightCycler and are recommended for product identification. When using sequence-specific probes, following an increase in fluorescence with cycle number can easily identify specific PCR products in the reaction mixture. The sequence-specific nature of the probes ensures that a fluorescence signal will only be obtained if the specific template is present in the reaction. Fluorescence monitoring of amplification using either hybridization or exonuclease probes is illustrated in Figure 5. The two probe systems have their own distinct artifacts: For exonuclease probes, the fluorescent signal increases with each cycle as the probe is hydrolyzed and the fluorophore is released from quenching. However, once amplification has reached the plateau phase the fluorescent signal continues to increase even when no net product is synthesized. This generates an artifactual increase in signal that is unrelated to product formation. For hybridization probes, the fluorescent signal also increases with cycle number but sometimes decreases in the plateau phase of the amplification. There are three possible explanations for this decrease: 1) Partial hydrolysis by the 5 to 3 exonuclease activity of Taq polymerase, 2) competition from reannealing product, 3) high product concentration favoring single over dual probe hybridization. Designing hybridization probes for product identification When designing hybridization probes for product identification a two-probe system is usually used. Probe-probe system. A two-probe system is recommended for product identification on the LightCycler. Both probes should be of similar length and Tm. Probe Tm should typically be higher than the primer Tm. The labeled probes should be designed so that when the probes are hybridized to the template, fluorophores are adjacent, allowing a FRET signal to be generated. This is typically achieved by labeling one probe at the 3 end and the other at the 5 end. Experimental Design - 18

19 Probe-probe system for Product Identification Template Probe A Probe B Monitoring Fluorescence Fluorescence is typically monitored at the end of a combined annealing/extension step to generate a fluorescence curve. Keep in mind that when hybridization probes are used to generate a FRET signal, fluorescence should be displayed as F2/ F1 or F2/1. The fluorescent signal will increase with cycle number as the specific product is amplified. For a detailed description of how to design and synthesize hybridization probes see Part 3.2 of this chapter. Examples of experiments that use sequence-specific probes for product analysis are described in detail in the Fluorescence Protocols section of this manual. 2.2 Principles of mutation detection using hybridization probes FRET hybridization probes The LightCycler is able to detect signals generated by fluorescence resonance energy transfer (FRET) between two adjacent fluorophores. As the two probes hybridize to the template a fluorescence signal from FRET is generated. As they dissociate from the template this signal is lost. The temperature at which a probe dissociates or melts away from the template is the apparent Tm of the probe. The same principles of product melting temperature can be applied to the melting temperature of probes off a long DNA template. The melting temperature of a hybridization probe is determined by its GC con- Experimental Design - 19

20 tent, length and sequence and whether it is perfectly matched to the template to which it is hybridized. Even a single base mismatch between probe and template significantly reduces the probe Tm Using FRET hybridization probes for mutation detection Single base mutation detection has been demonstrated on the LightCycler by amplification of a target sequence incorporating a fluorescently labeled primer. This generates a labeled strand of DNA to which a fluorescent probe is allowed to hybridize. Hybridization brings two fluorophores in close proximity, generating a FRET signal that is detected by the LightCycler. Raising the temperature of the reaction mixture causes the probe to melt away from the template at a characteristic temperature with a concomitant decrease in the fluorescence signal. The melting temperature of a probe can be reduced 5-10 C by a single base mismatch, causing a very well defined shift in melting peaks. Figure 6 illustrates a genotyping experiment for the factor V Leiden mutation using a labeled template and a single hybridization probe. In this experiment the hybridization probe is complementary to the wild type sequence. Notice how the probe melting temperature is lowered when paired with the mutant allele. A heterozygote is easily distinguished by the presence of two melting peaks representing both wild type and mutant alleles. Designing hybridization probes for mutation detection. When designing hybridization probes for mutation detection either a two probe or a primer-probe system can be used. Probe-probe system. If a two-probe system is selected for mutation detection one probe is used as a long anchor probe. A second shorter probe is then designed to lay over the mutation you are interested in (Figure 7B). In this way the Tm of the shorter probe always determines the fluorescence signal, so that any change in probe Tm caused by the presence of a mutation is reflected in the melting curve. The labeled probes are designed such that when the probes are hybridized to the template, fluorophores are adjacent, allowing a FRET signal to be generated. This is typically Experimental Design - 20

21 Figure 6. Factor V mutation detection. Cy5 ( Fluorescein ) 4 3 Homozygous Mutant Homozygous Wildtype Heterozygous Mutant No DNA Fluorescence df/dt Temperature ( C) achieved using adjacent probes and labeling one probe at the 3 end and the other at the 5 end. Primer-probe system. In this system, a fluorescently labeled primer is used to generate a labeled strand of template DNA. The concentration of labeled template can be increased by asymmetric amplification. A fluorescently labeled probe is designed such that hybridization of probe to labeled template brings two fluorophores into close contact (Figure 7A) generating a FRET signal detected by the LightCycler. This type of probe design can be used for mutation detection and product identification. Experimental Design - 21

22 Probes can be designed to be complimentary to either the wild type sequence or a mutant sequence. In the case of single base mutation detection it is helpful to have as large a difference in probe Tm between wild type and mutant sequences to clearly differentiate between the two alleles. Listed below are mismatched base pairs in order of decreasing stability: G-T > G-G = A-G > T-G > G-A = T-T > T-C > A-C > C-T Note: Even the most stable (G-T) mismatches have been used for mutation detection using the LightCycler, suggesting that any mismatch can be identified using this technique. In some cases it may be useful to design a probe that is complementary to the mutant sequence if the mutant probe-wild type template combination is less stable than pairing between the wild type probe and the mutant template. This can be used to maximize the Tm differences between the two alleles. For a detailed description of how to design and synthesize hybridization probes see Part 3.2 of this chapter. A typical protocol for single base mutation detection is described in detail in Section 5 of the Fluorescence Protocols section of this manual. Figure 7 A. Primer-probe system B. Probe-probe system Experimental Design - 22

23 2.3 Principles of Quantification using Fluorescence Fluorescence monitoring of PCR is a powerful tool for DNA quantification. Both double strand DNA (dsdna) dyes and sequence specific probes can be used for DNA quantification Setting up a quantification experiment Selecting standards The simplest approach to quantitative PCR is to measure the fluorescence of a PCR product in the log-linear phase of amplification with reference to the dilution series of an external standard. Quantification of PCR products using external standards has been demonstrated using the LightCycler and is described in the Fluorescence Protocols section of the manual (Sections 3A, 4A, 4B and Section 5). As an example, a section of the human b-globin gene was amplified and the product followed using either SYBR Green I (Section 3A) or hybridization probes (Section 4A and 4B). A known amount of a purified b-globin amplicon was used as the standard. There are several advantages to using external standards for quantification, notably the low complexity of the system. Some thought should go into designing the PCR products that will be used as standards. The simplest standards are purified PCR products; however, purified plasmid preparations are also commonly used. Any DNA products used as standards should be gel purified to remove traces of primers which may interfere with subsequent amplification. Standards used for quantification should be amplified under the same PCR conditions as the unknowns and have the same amplification efficiency. Amplification of standards and unknowns using the same primer set is advisable. The efficiency of amplification can be gauged using the LightCycler by comparing the cycle number at which the fluorescence signal appears out of the background and by the slope of the amplification curves. Products that amplify with the same efficiency have the same slope during the log-linear phase of amplification and, when present at the same concentration, should rise out of the background at the same cycle number. Experimental Design - 23

24 Determining copy number from fluorescence curve data If fluorescence is acquired each cycle, the LightCycler will generate a fluorescence vs cycle number curve for the data. The most useful data for quantification are fluorescence readings above the background when amplification is log-linear. The LightCycler quantification software is designed to generate a best-fit line for data within the log-linear portion of each curve. The point at which this line intercepts a defined fluorescence level identifies the crossing point or fractional cycle number that is related to the starting copy number of the sample. Quantitative data can be obtained by interpolating the fractional cycle number of an unknown sample against a standard curve of known concentrations (Figure 8). The LightCycler software requires you to define the log-linear part of the fluorescence curves displayed. This is done by setting thefluorescence level, or noise band on your screen to a level just above the background fluorescence. Then, the number of points that fall in a straight line above the noise band is set in the points box. This section of each curve is then fitted to a straight line that crosses the noise band. If you have entered a series of known standards along with your unknown samples, the LightCycler will plot a standard curve of concentration versus fractional cycle numbers and extrapolate values for the unknowns from this standard curve Selecting a fluorescence method for quantification Quantification with SYBR Green I Advantages of SYBR Green I Inexpensive Easy to use. Simply include diluted dye in the reaction as an additional reagent. Reactions can be monitored on the LightCycler without the synthesis of any fluorescently labeled probes. Disadvantages of SYBR Green I Sequence specificity is limited to primer specificity. Experimental Design - 24

25 Figure B 10 M Copies J 1 M Copies H 100 K Copies F 10 K Copies 1 K Copies 100 Copies B B B B B B B B B B B B B B B B B B B B B B B B B J J J J J J J J J J J J J J J J J J J H H H H H H H H H H H H H H H H F F F F F F F F F F F F F J H F J F H -0.3 B J F H B B J J H H F F F Noise Band Intersection of the Log-line with the Noise Band Cycle Number The generation of dsdna during PCR allows the study of DNA amplification products using dsdna dyes. These dyes are extremely useful because they can be used in any PCR reaction without the need for the synthesis of fluorescently labeled probes. SYBR Green I is particularly attractive because it has a high affinity for dsdna and produces a large increase in fluorescence upon binding. As PCR proceeds, the synthesis of new dsdna causes an increase in SYBR Green I fluorescence. Monitoring the amount of product each cycle, in a reaction containing SYBR Green I, allows analysis over a wide dynamic range of initial template concentration. Experimental Design - 25

26 Figure 9 illustrates the amplification of varying concentrations of a fragment of the beta globin gene. Quantification of initial template copy number is possible over a million-fold range. The fluorescence increases with cycle number in a sigmoidal fashion. The cycle number at which the fluorescence rises out of the background and increases in a log-linear manner can be used to determine the starting copy number of the template. Higher copy numbers cause the fluorescence threshold to be shifted to the left. Sensitivity, however, is limited at very low copy number because amplification specificity is not perfect. Because SYBR Green I binds to all dsdna, the presence of nonspecific products such as primer-dimers in the reaction also increases the fluorescence signal, especially at low template concentration. Figure 9 Fluorescence copies 1 copy 10 copies 100 copies 1,000 copies 10,000 copies 100,000 copies 1,000,000 copies Cycle Number Figure Specific Product -df/dt 0.8 Non-specific Product Acquire Here Temperature ( C) Experimental Design - 26

27 Increasing specificity with SYBR Green I If you decide to use SYBR Green I to follow your reaction, you can minimize the contribution of signal from nonspecific products using a combination of two techniques described below. Fluorescence can be acquired just before the melting temperature of your product (Figure 10). This recommendation is based on the fact that the Tm of primer-dimers and other small products is usually lower than the Tm of longer dsdna products. If fluorescence is acquired after these small products melted, they cannot contribute to the SYBR Green I fluorescence signal. In order to determine the melting temperatures of specific and nonspecific products you must generate a melting curve after amplification (see the Programming section of this manual for more details). Specific products tend to melt sharply at their Tm whereas nonspecific products usually generate broad peaks at lower temperatures. The specificity of the reaction can also be improved by including an anti-taq antibody in your reaction. This minimizes the formation of primer-dimers that may occur during preparation time. Anti-Taq antibodies are especially useful when quantifying low copy numbers. Using a combination of these two techniques SYBR Green I has been used to quantify gene targets from cdna with sensitivity down to the single copy level. Quantification with sequence-specific probes Advantages of sequence-specific probes Specificity. The fluorescent signal from labeled hybridization probes is directly related to product formation, without any contribution from nonspecific products. Disadvantages of sequence-specific probes A set of fluorescent probes must be designed for every product being monitored. Fluorescently labeled probes can be expensive to purchase or synthesize. Experimental Design - 27

28 Sequence-specific probes are often preferred when specificity is a priority. The fluorescence signal obtained from labeled probes can be a direct measure of product concentration. Sequence specific probes have an advantage over SYBR Green I because they offer enhanced specificity when the quantification of only a few copies of template per reaction is required. The use of fluorescently labeled probes for quantification requires the synthesis of a fluorescently labeled hybridization probe set and is, therefore, more complex than simply using a double strand DNA dye. Hybridization probes were specifically designed for the LightCycler. Designing hybridization probes for quantification. When designing hybridization probes for quantification either a two-probe or a primer-probe system can be used. Two-probe system. If a two-probe system is selected for quantification both probes should be of similar length and Tm. The labeled probes should be designed so that when the probes are hybridized to the template, fluorophores are adjacent, allowing a FRET signal to be generated. This is typically achieved by labeling one probe at the 3 end and the other at the 5 end. Primer-probe system. A fluorescently labeled primer can be used to generate a labeled strand of template DNA. A fluorescently labeled probe should be designed so that hybridization of probe to labeled template brings two fluorophores into close contact generating a FRET signal detected by the LightCycler. Monitoring Fluorescence Fluorescence is typically monitored each cycle at the end of the annealing step to generate a fluorescence curve. Keep in mind that when hybridization probes are used to generate a FRET signal fluorescence should be displayed as F2/F1 or F2/1. Quantification of starting copy number is possible by comparing the crossing point or fractional cycle number of the unknown sample to the standard curve made with known amounts of purified amplicon. For a description of the LightCycler quantification software please read the Programming section of this manual. Examples of experiments that include quantification analysis are described in detail in the Fluorescence Protocols section of this manual. Experimental Design - 28

29 PART 3 FLUORESCENCE MONITORING SYSTEMS 3.1 Using SYBR Green I with the LightCycler Advantages of SYBR Green I Flexibility. SYBR Green I recognizes all dsdna regardless of sequence, therefore no additional reagents except for the dye are required in order to monitor the accumulation of amplification products. Cost. SYBR Green I is recommended as the dsdna dye of choice for the LightCycler, this dye is easily available, affordable and is used at a high dilution. Disadvantages of SYBR Green I Limited specificity. If PCR amplification is highly optimized, dsdna dyes such as SYBR Green I are highly recommended. However, because these dyes recognize all dsdna, the presence of nonspecific products or contaminants can cause an increase in fluorescence not related to the target DNA. This drawback can be minimized with the LightCycler by using melting curves to eliminate the signal from many, but not all, nonspecific products. Experimental Design - 29

30 SYBR Green I is particularly suited for use with the LightCycler It is compatible with PCR at concentrations that give a strong fluorescence signal. It binds in the minor groove of dsdna with minimum sequence selectivity. It is stable under rapid temperature cycling conditions. The maximum excitation wavelength of SYBR Green I is 497 nm allowing good excitation by the blue LED of the LightCycler. The LightCycler is also designed to detect fluorescence at the maximum emission wavelength of SYBR Green I at 520 nm (F1 channel). Although SYBR Green I is extremely useful for monitoring the behavior of DNA with respect to temperature, it should be noted that there are some nonspecific effects of temperature on the fluorescence of the dye as shown in Figure 11. Highest fluorescence values are obtained at lower temperatures. Figure Temperature ( C) Fluorescence Time (sec) Experimental Design - 30

31 Recommended concentrations of SYBR Green I SYBR Green I is available from Roche Molecular Biochemicals in an optimized formulation ready-to-use in the LightCycler. No molecular weight for SYBR Green I is available. A typical 10X working solution is usually a 1:3000 or even a 1:6000 dilution of the stock (final concentration 1:30,000 or 1:60, 000). Note: If you purchase SYBR Green I from Idaho Technology, it is shipped as a 1:1000 dilution of the original Molecular Probes stock and should be diluted at 1:3 or 1:6 to obtain a 10X working solution. Influence of Magnesium ion concentration on SYBR Green I fluorescence The concentration of magnesium in your reaction mix can influence the fluorescence of SYBR Green I. High concentrations of magnesium will decrease the SYBR Green I fluorescence signal (Figure 12). Most PCR reactions are optimized to work at magnesium concentrations between 1 and 5 mm. If your reaction is optimized for high concentrations of magnesium (4 mm or higher) keep in mind that the intensity of your SYBR Green I fluorescence signal will be decreased. This is also important when comparing multiple reactions containing different concentrations of magnesium. Figure 12. Effect of Mg. Fluorescence mm 2 mm mm mm 5 mm 0 mm Temperature ( C) Experimental Design - 31

32 3.2 Using hybridization probes with the LightCycler The use of hybridization probes in DNA analysis is described in the introduction to this manual. Briefly, fluorescence resonance energy transfer (FRET) can occur between two fluorophores placed on hybridization probes that anneal to the target DNA in close proximity. The LightCycler monitors this FRET signal. The amount of FRET signal is related to the amount of specific DNA product allowing product identification, mutation detection, and quantification. Advantages of hybridization probes Specificity. In contrast to SYBR Green I and other dsdna dyes, hybridization probes provide complete sequence specificity. Only those DNA sequences recognized by the probes are involved in generating a FRET signal, eliminating the fluorescence signal generated by nonspecific products. Mutation detection. A unique strength of hybridization probes is the ability to use these probes for mutation detection, including single base alterations. Disadvantages of hybridization probes Lack of flexibility. Unlike dsdna dyes, each fluorescence experiment involving hybridization probes requires the synthesis of specific labeled probes designed with a particular target sequence in mind. Cost. The use of hybridization probes in LightCycler experiments requires the synthesis of fluorescently labeled probes, which is significantly more expensive than using dsdna dyes. Experimental Design - 32

33 3.21 The design and synthesis of fluorescently labeled oligonucleotides Design of adjacent Hybridization probes. Follow the guidelines below when designing hybridization probes: Positions of probes on target DNA. In general it is recommended that both probes should be placed on the same strand near to, but not overlapping the primer on the opposite strand. This allows time for hybridization before the probes are displaced by extension. However, this type of probe design can be restricted by the size of the amplicon. Probes immediately adjacent to a primer have also been used in LightCycler experiments with success. Primer 1 Probe 1 Probe 2 Primer 2 Probe length. For best results probe length should be between 23 and 30 base pairs. Probe sequences. If used for quantification or product identification, the melting temperature of the two hybridization probes should be nearly equal. If used for mutation detection it is recommended that one probe be longer (an anchor probe) and that a shorter probe is designed to lie over the predicted mutation site. This ensures that the Tm of the shorter probe is lower than the Tm of the anchor probe so that only the probe that sits over the mutation site determines the fluorescence signal. Remember, probe Tm is dependent on both length and GC content, the latter being more significant, so careful selection of probe sequences is important. The Tm of your probes should be 5-10 C higher than the Tm of your amplification primers. Experimental Design - 33

34 Block the 3 end of your hybridization probes. Hybridization probes MUST be blocked from extending so that they do not compete with the amplification primers. If you design a probe with FITC on the 3 end this will automatically block extension from this probe. For probes that have either FITC or Cy5 on the 5 end, the 3 end must be blocked with a phosphate group. This can be done during synthesis. The optimum separation between FRET dyes is one base pair. A one base separation between fluors gives the maximum change in fluorescence ratio. However, if necessary, a distance of up to six bases between the two dyes will work well for most applications. Fluorescence signal will decrease with fluor separation of greater than ten bases. Select the concentration of probe. The ratio of Cy5 labeled probe to FITC labeled probe must be optimized for each probe set. Maximum FRET signal is usually obtained at a Cy5-probe to FITC-probe ratio between 1:1 and 4:1. Try a Cy5 to FITC ratio of 2:1 at a fluorescein concentration of 0.2 µm and a Cy5 probe concentration of 0.4 µm. These concentrations can be modified by a factor of 2-4 for optimal results. Optimize your reaction. LightCycler reactions can be optimized for several factors, for details see the Protocols section of this manual. Some important considerations when designing reactions using hybridization probes are described below: Optimize your reactions for Mg 2+ concentrations. Try using Mg 2+ concentrations between 1 and 5 mm to determine the best concentration for your amplification reaction. Hybridization probe reactions often work better using two-temperature cycling conditions, the combined annealing extension step allows more time for probe hybridization. For example, set up a denaturation segment at 94 C for 0 seconds followed by annealing / extension steps at 58 C, 60 C, 62 C, and 64 C. Hold this annealing / extension step for 20 seconds. This protocol usually works well for products between 100 to 400 base pairs in length. Experimental Design - 34

35 Set up your program so that the fluorescence acquisition occurs at the end of the combined annealing / extension step in each cycle. This will allow you to monitor the increase in your target DNA concentration as the amplification proceeds. For details of how to include this acquisition step in your program see the Programming section of this manual. Remember that the FRET signal is mainly from the acceptor fluor, so the fluorescence display must be set at F2/F1 or F2/1. As the PCR reaction proceeds, the FRET signal increases until it reaches an amplification plateau. Optimize your reaction so signal appears out of the background at the earliest cycle number possible and the greatest number of log-linear points is obtained. Synthesis of Adjacent Hybridization Probes. Fluorescently labeled hybridization probes can be either synthesized by the investigator or purchased commercially. The following section contains guidelines for probe synthesis. These guidelines are targeted at investigators who are interested in synthesizing their own fluorescently labeled probes and some familiarity with oligonucleotide synthesis is assumed. Synthesis of a 3 -Fluorescein labeled probe. Use a fluorescein-labeled CPG cassette (Glen Research or Biogenix) Synthesize the probe with trityl-on Remove the failure sequences and the trityl group on a Polypack (Glen Research). Elute with 50% acetonitrile. Confirm the purity of the probe by reverse phase HPLC (monitored by both A260 absorbance and Ex490 nm, Ex520 nm and fluorescence) and purify by HPLC if necessary. Synthesis of a LC Red 640 labeled probe. Add a 3 phosphate and a 5 amino linker during synthesis. Synthesize the probe with trityl-on Remove the failure sequences and the trityl group on a Polypack (Glen Research). Experimental Design - 35