Supplementary Methods

Transcription

1 Supplementary Methods SMAP Assay Detailed Protocol I. Technology Description SMAP is a novel gene amplification method with high sensitivity and single-nucleotide precision, enabling rapid detection of SNPs, mutations, and other DNA targets. The method employs two unique approaches to suppress mis-match (background) amplification including a unique primer design and enzyme combination. SMAP is an isothermal DNA amplification method that yields copious amounts of amplified product (~100x greater than PCR). When SMAP is optimized, complete suppression of background can be achieved and hence, amplification becomes equivalent to detection. AMPLIFICATION = DETECTION is the basic principle of SMAP. SMAP can be used for rapid amplification of Single Nucleotide Polymorphism (SNP) genotyping, mutation detection, or other molecular diagnostics applications. Samples such as blood, cultured cells, tissue samples, or purified DNA can be analyzed. The SMAP assay is performed at 60 C for at least minutes depending on the application and primer set, and results are usually read on Real-time PCR instrumentation. II. SMAP Assay Reagent Requirements A description of the materials and components for assembly of a SMAP assay are given in Table 1 (below). The nomenclature (Labels) and design of the components are compatible with the detail protocol and figures described in these Supplementary Methods. Table 1. SMAP Assay Reagents Description Labels Contents (1) Specimen Pretreatment Solution PS 50 mm NaOH (2) Water DW DNA, RNA, Nuclease-free (3) 2X Reaction Mix RM 28 mm dntps, 10% DMSO, 40 mm Tris HCl (ph8.8), 20 mm KCl, 20 mm (NH4)2SO4, 16 mm MgSO4, 0.2% Tween 20, 1/50,000 dilution of original SYBR Green I (Invitrogen ) (4) Aac DNA polymerase Aac Available from DNAFORM K.K. (5) Thermus aquaticus Taq Mutase MutS Available from DNAFORM K.K. (6) Positive Control DNA Template PCT 1x Plasmid Control DNA (~6,000 (ALDH2) copies /μl plasmid DNA in water) (7) Positive Control DNA Primers (ALDH2) PCP 2.5μM OP1, 2.5μM OP2, 20μM TP, 20μM FP, 10μM BP in TE 1

2 III. SMAP Assay Assembly Considerations Each SMAP reaction for detecting a specific SNP sequence should be performed independently, and in separate tubes. Generally, each tube will contain a different SMAP primer mix, one for amplification of the wild-type target and one or more for detection of various specific SNP forms or mutations. Additionally, three control tubes are recommended; a positive control for genomic DNA integrity, a positive control for checking reagent integrity (plasmid target), and a negative control for monitoring contamination (no genomic DNA). Interchelating fluorescent dyes are added to the reaction mix, and fluorescence intensity is measured with Real-time thermocycling (PCR) instruments. SMAP is an isothermal reaction, so the amplification occurs at constant temperature and is time dependent (no thermocycling is required). In an optimized SMAP assay, full mis-match suppression can be achieved, and data analysis essentially becomes digital in nature; amplification is either positive or negative. IV. Specimen Preparation Regardless of the specimen type (blood, tissue, cells, or purified DNA), the optimal SMAP assay for real-time instrument detection requires the equivalence of ~6,000 target gene copies. The SMAP reaction is very robust and specificity usually will not be affected by 10 to 50-fold over-estimation or under-estimation of target copies. Interpretation of results will not be affected with DNA load variance within this range even through amplification time will vary (inverse relationship). This protocol describes how to configure a SMAP assay in a 25μL total volume, with 1μL contributed by the pretreated sample. The specimen pretreatment solution (50 mm NaOH) can be used for preparation of blood, tissue, or cultured cells (use water for purified genomic DNA). For each kind of specimen, a brief heat treatment is required to ensure cell lysis and denaturation of the DNA. This treated sample (1μL) can then be directly assayed in the SMAP reaction. Blood Speciman Preparation. Each SMAP reaction requires less than 1μL of whole blood per assay. To prepare a single assay, mix 1μL of whole blood with 2μL of the pretreatment solution (PS). When performing multiple assays, scale-up your volume requirements accordingly. Heat the mixture at 98 C for 3 minutes, vortex, and quench on ice. Use 1μL of the sample preparation directly in a SMAP assay. Purified Human Genomic DNA. For assaying human genomic DNA, the sample should be diluted to a concentration of 40 ng/μl, which corresponds to the equivalence of approximately 3,000 human (diploid) cells in one microliter. Prior to performing the SMAP assay, heat the mixture at 98 C for 3 minutes, vortex, and quench on ice. Use 1μL of the sample preparation directly in a SMAP assay. 2

3 Positive Control DNA (plasmid) & Control Primer Set. It is advisable to create a plasmid positive control template (PCT) and positive control primer set (PCP) for aiding in the design and troubleshooting of SMAP assays. For this purpose, the ALDH2 gene locus and primer set is suitable. The PCT should be a clone of the wild-type human ALDH2 locus prepared in water or TE at a concentration of approximately 6,000 molecules per microliter. The PCP should be 10X concentration, which is 32 μm each of FP and TP, 4 μm each of OP1 and OP2, and 16 μm BP (Table 4). At least one SMAP reaction using the control DNA and control primers should be run for each experiment to serve as a check on the integrity of the SMAP reagents and buffers. To prepare a single plasmid control reaction, aliquot a small volume (at least 5μl) of PCT and heat the mixture at 98 C for 3 minutes, vortex, and quench on ice. Assembly of a 25μl SMAP assay requires 1μl of PCT be added into the Positive Control Master Mix (PCMM) to assemble the Reagent Positive control (RPC) SMAP assay (Fig. 1). The PCMM is prepared similar to all other Master Mix, as described in Table 3. Alternative Positive Control (human genomic target DNA). Most of the time, the control primer set can also amplify the target gene sequence from your prepared human specimen. The ALDH2 primer set will efficiently amplify the human allele within a conserved region. Although one SNP variant is known to exist in this region, the frequency of a homozygous mutant in the human population is rare. If your target DNA is a homozygous mutant, then it will not amplify. Performing this control reaction is optional, but it will tell you if the integrity of your genomic DNA preparation is sufficient for achieving good SMAP results with good primers. Mix 1μl of the specimen preparation with your Positive Control Master Mix (PCMM) to assemble the Specimen Positive Control (SPC) SMAP assay. You may also want to set up assay tubes SPC1, SPC2, SPC3, etc., to validate the integrity of the genomic DNA of all test samples (optional). Negative (Contamination) Control. A control for monitoring contamination should be performed with each SMAP experiment. This control consists of a Master Mix plus 1μl of water (no template DNA). If there is contamination in the Pre-Mix or the primer set specific for that Master Mix, it will be detected (amplification will result). At least one negative control assay (NC) should always be performed to be sure of the Pre-Mix integrity. One negative control for each Master Mix (NC1, NC2, NC3, etc.) can be performed to thoroughly monitor all primer sets for contamination, as well. 3

4 V. SMAP Assay Preparation A) Pre-Mix Reagent Preparation 1) Depending on the number of specimens and primer sets to be tested, calculate the volume needs for assembling the Pre-Mix and Master Mixes. Consult Figure 1 for determining the total number of assays to be performed on your samples. The SMAP Pre-Mix is used for all assays and control reactions; scale-up the volumes proportionately. As shown in the table given, each reaction will require a volume of 21.5μL of Pre-mix per SMAP assay. 2) The Pre-Mix should be assembled on ice, according to Table 2, in a sterile microfuge tube or equivalent container (depending on volume). The volumes of Aac DNA Polymerase and Taq MutS may vary depending on the primer set employed. If you or a colleague designed the primer set, use the optimal conditions that were previously determined. Otherwise, you may want to use 1μL of Aac DNA Polymerase titrate MutS to optimize for the minimal amount required to suppress any mis-amplification. 4

5 Prepare the required volumes. An additional reaction, or an additional 10% is recommended to account for pipeting variance. Table 2. Pre-mix preparation (1 reaction) Reagent Amount Distilled Water (DW) μl (typically 6.0μl) Reaction Mix (RM) 12.5 μl Aac DNA Polymerase (Aac) μl (typically 1.0μl) Taq Mutase (TaqMutS) μl (typically 2.0μl) Total 21.5 μl 3) Agitate the Pre-Mix no more than 5 times for 1 second each with a vortex mixer. Centrifuge for a few seconds to collect the components to the bottom of the tube. Please note that too much mixing with the vortex mixer may inactivate (denature) the enzymes. B) Master Mix Reagent Preparation 1) A different primer set is required for detection of each SNP or mutation (or independent target). You will need to prepare a Master Mix for each primer set. Into separate sterilized 1.5mL microfuge tubes, dispense the appropriate amount of Pre-Mix and the specific SMAP primer mix, according to the volumes recommended in Table 3 (below). Scale-up your volume requirements as needed. In each Master Mix preparation, be sure to include enough for one negative control (NC) and one reagent positive control (RPC) to check for contamination of reagents and SMAP reagent integrity, respectively. The alternative specimen positive control (SPC) may also be included to check on template integrity for one or all specimens. Table 3. Master Mix preparation (1 reaction) Reagent Amount Pre-mix 21.5 μl Primer Set μl Total 24.5 μl 2) Agitate the master mix no more than 5 times for 1 second each with a vortex mixer. Centrifuge for a few seconds to collect the components to the bottom of the tube. Please note that too much mixing with the vortex mixer may inactivate (denature) the enzymes. Use the prepared Master Mix immediately. Do not let sit on ice for an extended period of time. 5

6 Prepare the required volumes. An additional reaction, or an additional 10% is recommended to account for pipeting variance. C) Assay Final Assembly You will need to assemble the assays in the reaction tubes that are compatible with your detection instrumentation. Set-up reactions on wet ice. 1) To each specimen assay tube, add 24.0μL Master Mix and 1.0μL of pretreated specimen that was previously prepared. To the Reagent Positive Control (RPC) tube, use 1μL of the Positive Control Template (PCT) that was previously prepared. To the Specimen Positive Control (SPC) tube(s), use 1μL of the Specimen template that was also previously prepared. For the negative control(s), use 1μL of the distilled water (DW). 2) Mix the solutions no more than 5 times for 1 second each with a vortex mixer. Centrifuge for a few seconds to collect the components to the bottom of the tube. Please note that too much mixing with the vortex mixer may inactivate (denature) the enzymes. VI. Amplification reaction and Instrumentation Settings. Set up your Real-time PCR instrument with excitation and emission detection wavelengths compatible with the interchelating dye you choose to use. We ve had good results with both SYBR Green I (Invitrogen ), and EvaGreen (Biotium ). Any Real-time PCR instrument is probably acceptable for SMAP assays. Set parameters to measure the fluorescence intensity in every assay tube at 1 minute intervals. Incubate the reaction tubes at a constant temperature of 60 C for minutes, or as appropriate for your primer set. VII. Interpretation of Results (Real-time Analysis) The principle of SMAP involves suppression of mismatch amplification. But depending upon one s skill at designing SMAP primers and assays, suppression may mean short delay mismatch amplification, long delay mismatch amplification, or total suppression of mismatch amplification (Fig.2). You should know your primer set characteristics before conducting actual clinical (diagnostic) experiments. Controlled experiments should be run on your primer set to determine at what detection time point mismatch amplification begins to appear (if at all). For best results, use cloned wild-type and mutant templates (purified plasmid) for controlled SMAP experiments. If the time period between full-match amplification and mismatch amplification is too short (less than min), your 6

7 primer set generally still requires sequence design optimization, or more Taq MutS in the SMAP reaction, or both. Each primer set should be checked against each template to verify specificity and mismatch amplification delay time. In isothermal amplification reactions like SMAP, amplification time is inversely proportional to template load. If for example, you have demonstrated that full-match amplification is evident at 30 minutes, and mismatch amplification does not appear until 1 hour (or not at all), then your detection time (Dt) should be set at minutes (or thereabout). Endpoint determination assays can only be designed with primer sets that display full suppression of mismatch amplification. See figure 2 (below) for a visual illustration of these concepts. If primer sets require optimization, consult the SMAP server for recommendations regarding SMAP primer rational design. SMAP results can be analyzed qualitatively or quantitatively. At the proper detection time (Dt) for your optimized assay, data should be collected and analyzed. At this time point, there will be mismatch suppression, while positive amplification will yield copious amounts of DNA. Hence, the interpretation of results is usually unmistakable. The results can be viewed as digital; positive or negative. The presence of a SNP, sequence, or mutation is confirmed if amplification is observed in an assay tube that uses a primer set specific for that target. The SNP, sequence, or mutation is NOT present if no amplification is observed in the tube. 7

8 VIII. SMAP Primer Sets Table 4. ALDH2 Primer Set for SMAP (human aldehyde dehyrdrogenase 2) TP 5 -CGAGTACGGGCCCACACTCACAGTTTTCAC-3 FP 5 -TTTATATATATATAAACCGGGAGTTGGGCGAG-3 BP (wild-type) 5 -GCAGGCATACACTGA-3 BP (mutant) 5 -GCAGGCATACACTAA -3 OP1 5 -ACAAGATGTCGGGGAGTG-3 OP2 5 -CCTGAGCCCCCAGCAGGT-3 Table 5. CYP2C19*2 Primer Set for SMAP (human cytochrome P450 type 2C19) TP (wild-type) 5 -CCCGGGAAATAATCATAAATTATTGTTTTCTCTTAGATA -3 TP (mutant) 5 -CCTGGGAAATAATCATAAATTATTGTTTTCTCTTAGATA-3 FP 5 -CCTATATATATATAGGAGGTTTTTAAGTAATTTGTTATGGG-3 BP 5 -TGATAGTGGGAAAATTATTG-3 OP1 5 -TATTAAATGCTTTTAATT-3 OP2 5 -TCGATTCTTGGTGTTCTT-3 Table 6. DIO2 Primer Set for SMAP (human type 2 iodothyronine deiodinase) TP (wild-type) 5 -TACTGGAGACGTGAAATTGGGTGAGGATGC-3 TP (mutant) 5 -TGCACTGGAGAGTGAAATTGGGTGAGGATGC-3 FP 5 -TTTATATATATATAAAGTACCATTGCCACTGTT-3 BP 5 -CACACTGGAATTGGGGG-3 OP1 5 -TCAGCTATCTTCTCCTGG-3 OP2 5 -TGTGATATTCTCACCTTC-3 Table 7. ADRB3 Primer Set for SMAP (human β3 adrenergic receptor) TP (wild-type) 5 -TGGACTCCCAGCGAAGTCACGAACACG-3 TP (mutant) 5 -CGGACTCCCAGCGAAGTCACGAACACG-3 FP 5 -GCGAGTCGCTCATCGTGGCCATCG-3 BP 5 -CAGACCATGACCAA-3 OP1 5 -TGGGAGGCAACCTGCTGG-3 OP2 5 -GTCCCATCACCAGGTCGG-3 IX. URLs Table 8. URLs for Additional Information SMAP server DNAFORM K.K. Invitrogen Biotium 8