USB HotStart-IT. for increased specificity and consistent results. PCR, qpcr and qrt-pcr

Transcription

1 USB HotStart-IT for increased specificity and consistent results PCR, qpcr and qrt-pcr

2 USB PCR Reagents Choose USB HotStart-IT products for increased specificity and consistent results. Long and Accurate PCR High Fidelity HotStart-IT FideliTaq TM DNA Polymerase [71155] HotStart-IT FideliTaq TM PCR Master Mix (2X) [71156] Hot-Start PCR Novel Primer Binding HotStart-IT Taq DNA Polymerase [71195] HotStart-IT Taq PCR Master Mix (2X) [71196] HotStart-IT Binding Protein [71194] qpcr Master Mixes HotStart-IT SYBR Green qpcr Master Mix (2X) [75762] HotStart-IT Probe qpcr Master Mix (2X) [75766] HotStart-IT SYBR Green qpcr Master Mix with UDG (2X) [75760] HotStart-IT Probe qpcr Master Mix with UDG (2X) [75764] qrt-pcr Kits HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit [75770] HotStart-IT Probe One-Step qrt-pcr Master Mix Kit [75772] First-Strand cdna Synthesis Kit for Real-Time PCR [75780] PCR Components Nucleotides 10mM PCR Nucleotide Mix [77212] 25mM PCR Nucleotide Mix [77119] 10mM PCR Nucleotide Mix with dutp [77330] Purification DNA PrepEase Tissue & Cells DNA Spin Kit [78860] PrepEase Genomic DNA Isolation Kit [78850] PrepEase MiniSpin Plasmid Kit [78735] Reagents Nuclease-Free Water [71786] 5M Betaine Solution [77507] Reaction Buffers 10X PCR Reaction Buffer with MgCl 2 [71165] 10X PCR Reaction Buffer without MgCl 2 [71166] Passive Reference Dyes Fluorescein Passive Reference Dye [75767] ROX Passive Reference Dye [75768] RNA PrepEase RNA Spin Kit [78765] PrepEase Plant RNA Spin Kit [78770] PrepEase mrna MiniSpin Kit [78878] PCR Products ExoSAP-IT For PCR Product Clean-Up [78200] PrepEase DNA Clean-Up Kit [78758] PrepEase Gel Extraction Kit [78755]

3 USB Hot Start PCR Technology USB HotStart-IT provides researchers with a complete line of robust, sensitive and specific reagents for hot start PCR, qpcr, and qrt-pcr. Rely on USB HotStart-IT to provide premium quality and consistency for your most critical and routine PCR applications. Benefits High Specificity Avoids non-specific products and primer-dimer formation Convenient Room temperature reaction set-up Robust Withstands freeze-thaw cycles and storage for one month at room temperature. USB products having the Tested User Friendly TM brand are functionally tested in a specific application with a buffer and protocol. The tested buffer and protocol accompany each product, providing time-saving convenience and reducing the number of variables which could hinder your most critical experiments. Many TUF qualified enzymes are supplied with a reaction buffer and functionally tested enzyme dilution buffer. How It Works HotStart-IT Taq DNA Polymerase uses a novel hot start method called primer sequestration. In general, hot start PCR methods reduce or eliminate non-specific primer-extension products formed at lower temperatures during assembly of PCR reactions. At these less stringent annealing temperatures, primers can bind nonspecifically, which often leads to unwanted amplification products and primer-dimers. USB HotStart-IT uses a recombinant protein which binds and sequesters primers at lower temperatures making them unavailable for use by Taq DNA Polymerase. This primer-sequestration technique effectively blocks DNA synthesis from mispriming events at lower temperatures. Following the initial denaturing step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. This novel hot start method enhances PCR reactions by increasing both specificity and yield. USB HotStart-IT Method: Primer Sequestration

4 Hot Start PCR USB HotStart-IT Taq DNA Polymerase includes a recombinant protein which binds and sequesters primers at lower temperatures, making them unavailable for use by Taq DNA Polymerase. This primer-sequestration technique effectively blocks DNA synthesis from mispriming events at lower temperatures. Following the initial denaturing step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. High specificity and sensitivity Minimizes amplification of non-specific products and primer-dimers Room temperature reaction set-up HotStart-IT Taq DNA Polymerase units $ units $ units $ x 250 units $ units $2, HotStart-IT Taq DNA Polymerase Increased specificity of HotStart-IT Taq DNA Polymerase. The single-copy numb gene was amplified from the indicated amounts of human genomic DNA with standard Taq DNA Polymerase ( ) or with HotStart-IT Taq DNA Polymerase (+). Primers were designed with a 3 bp overlap at their 3'-ends to favor primer-dimer formation during reaction set-up at room temperature. Results demonstrate a shift from mainly primer-dimers to the desired product when HotStart-IT Taq DNA Polymerase is used. HotStart-IT Taq Master Mix (2X) HotStart-IT Taq Master Mix (2X) combines highquality USB recombinant Taq DNA Polymerase, a recombinant hot start protein, and USB Ultrapure nucleotides in a proprietary reaction buffer. This ready-to-use mix provides robust and reliable performance for demanding PCR applications in which high specificity and high sensitivity are desired. Since the mix is pre-formulated, experimental variability is significantly reduced. High specificity and sensitivity Robust mix withstands freeze-thaw cycles and storage for one month at room temperature Room temperature reaction set-up HotStart-IT Taq Master Mix (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ Increased specificity and stability of HotStart-IT Taq Master Mix. Specificity: The single-copy numb gene was amplified from 1 ng of human genomic DNA with standard Taq Master Mix ( ) or with HotStart-IT Taq Master Mix (+). Primers were designed with a 3 bp overlap at their 3'-ends to favor primer-dimer formation during reaction set-up at room temperature. Results demonstrate a shift from mainly primer-dimers to the desired product when HotStart-IT Taq Master Mix is used. Stability: The Master Mix shows no loss in performance following 15 freeze-thaw cycles (F/T), 4 C storage for one month, or room temperature storage for one month (RT) compared to a reference mix stored at 20 C. HotStart-IT Binding Protein USB also offers the HotStart-IT Binding Protein to give researchers the specificity and sensitivity of hot start PCR while also having the flexibility to use any desired PCR polymerase. The HotStart-IT Binding Protein binds and sequesters primers at lower temperatures making them unavailable for use by the polymerase. Following the initial denaturing step during PCR, the protein is inactivated and the primers are free to participate in the amplification reaction. Choose your own polymerase and achieve a hot start PCR by supplementing the reaction mix with USB s HotStart-IT Binding Protein Enables room temperature reaction set-up HotStart-IT Binding Protein µg $88.00

5 Long and Accurate PCR HotStart-IT FideliTaq TM DNA Polymerase HotStart-IT FideliTaq TM DNA Polymerase combines HotStart-IT technology with the long and accurate amplification properties of FideliTaq TM DNA Polymerase. High specificity and sensitivity 6X increased accuracy compared to Taq DNA Polymerase Generate amplicons as large as 35 kb HotStart-IT FideliTaq TM DNA Polymerase units $ units $ units $ units $2, Extreme amplicon length using HotStart-IT FideliTaq TM DNA Polymerase. Two long PCR products of 20.7 kb and 35 kb were amplified from lambda DNA using 1 ng and 10 ng of DNA respectively. Cycling conditions consisted of a short initial denaturation phase at 95 C for 20 seconds and 25 cycles of 95 C for 5 seconds and 68 C for 20 minutes. HotStart-IT FideliTaq TM PCR Master Mix (2X) HotStart-IT FideliTaq TM PCR Master Mix (2X) combines HotStart-IT technology with the long and accurate amplification properties of FideliTaq TM DNA Polymerase in a convenient master mix. High specificity and sensitivity can amplify from single cell starting samples 6X increased accuracy compared to Taq DNA Polymerase Generate amplicons as large as 35 kb Convenience only need to add primers, template and water HotStart-IT FideliTaq TM PCR Master Mix (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ Sensitivity of the HotStart-IT FideliTaq TM PCR Master Mix. A 455 bp fragment of the single-copy numb gene was amplified from the indicated amounts of human genomic DNA. The master mix is extremely sensitive as amplification can be achieved from approximately one human cell.

6 qpcr HotStart-IT SYBR Green qpcr Master Mix is supplied as a 2X premixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl 2, Ultrapure nucleotides, and SYBR Green I for use in real-time quantitative PCR reactions (qpcr). The SYBR Green I dye detects any double-stranded DNA that accumulates during the amplification process. The hot start feature increases sensitivity and specificity of SYBR -based qpcr by reducing the formation of primer-dimers and non-specific products. Simply add DNA template, primers, and water and the reactions are ready for cycling. Separate tubes of the HotStart-IT SYBR Green qpcr Master Mix (2X) passive reference dyes, ROX and fluorescein, are included for added convenience. High sensitivity and consistency over a broad dynamic range Detects fewer than 10 target copies Performs over a linear dynamic range of 7 to 8 orders of magnitude with a minimal correlation coefficient 0.95 Room temperature reaction setup Robust master mix withstands repeated freezethaws with no detectable loss of efficacy HotStart-IT SYBR Green qpcr Master Mix (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ Real-time PCR from 10 8 copies down to a single copy target using HotStart-IT SYBR Green qpcr Master Mix R 2 = GAPDH Assay using HotStart-IT SYBR Green qpcr Master Mix (PN 75762). Triplicate reactions were performed with a cloned region of the human GAPDH gene as template using an ABI 7500 Fast instrument. The non-specific dsdna binding dye, SYBR Green I, was used to detect the 122 bp amplicon and ROX TM was used as a passive reference dye. The amplification process was linear over eight orders of magnitude and a single copy of the target could be efficiently detected. HotStart-IT Probe qpcr Master Mix (2X) HotStart-IT Probe qpcr Master Mix is supplied as a 2X premixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl 2, and Ultrapure nucleotides for use in real-time quantitative PCR reactions (qpcr) with fluorescent probes. Since fluorescent probes are designed to hybridize to the target of interest, detection specificity is greatly increased relative to non-specific dsdna binding dyes such as SYBR Green I. The Taq DNA Polymerase used in the master mix has the 5' 3' exonuclease activity necessary for efficient removal of the 5'-fluorophore from the 3'-quencher in TaqMan probes. Simply add DNA template, primers, probe(s) and water and the reactions are ready for cycling. A separate tube of ROX TM Passive Reference Dye is included for added convenience. High sensitivity and consistency over a broad dynamic range Detects fewer than 10 target copies Performs over a linear dynamic range of 7 to 8 orders of magnitude with a minimal correlation coefficient 0.95 Room temperature reaction setup Robust master mix withstands repeated freezethaws with no detectable loss of efficacy HotStart-IT Probe qpcr Master Mix (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ Real-time PCR from 10 8 copies down to a single copy target using HotStart-IT Probe qpcr Master Mix R 2 = GAPDH Assay using HotStart-IT Probe qpcr Master Mix (PN 75766). Triplicate reactions were performed with a cloned region of the human GAPDH gene as template using an ABI 7500 Fast instrument. A TaqMan probe with FAM as the reporter fluorophore and BHQ-1 as the quencher was used to detect the 122 bp amplicon. ROX TM was used as a passive reference dye. The amplification process was linear over eight orders of magnitude and a single copy of the target could be efficiently detected.

7 qpcr with carryover prevention HotStart-IT qpcr Master Mixes with UDG are supplied as 2X pre-mixed formulations containing HotStart-IT Taq DNA Polymerase, MgCl 2, Ultrapure nucleotides with an optimized dutp to dttp ratio, and heat-labile UDG for use in real-time quantitative PCR reactions (qpcr) with either fluorescent probes or SYBR Green Dye. Since the mix contains dutp and UDG, carry-over contamination prevention can be performed, which is especially important for high throughput applications. (1) The dutp in the mix ensures that products which contain uracil are destroyed prior to subsequent amplification reactions by the enzymatic activity of the Uracil-DNA Glycosylase (UDG). A heat-labile version of UDG that is irreversibly heat-inactivated is used instead of E. coli UDG, which has been shown to exhibit residual activity following PCR reactions. (2) After the initial denaturating step, the UDG is inactivated, and only the desired target sequences without dutp are amplified. Simply add DNA template, primers, probe(s) and water and the reactions are ready for cycling. A separate tube of ROX TM Passive Reference Dye and/or fluorescein is included for added convenience. References 1. Longo, M. C., Berninger, M. S., and Hartley, J. L. (1990) Gene 93, Thornton, C. G., Hartley, J. L., and Rashtchian, A. (1992) BioTechniques 13, HotStart-IT SYBR Green qpcr Master Mix with UDG (2X) HotStart-IT SYBR Green qpcr Master Mix with UDG is supplied as a 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase, heatlabile UDG, MgCl 2, Ultrapure nucleotides, and SYBR Green I for use in real-time quantitative PCR reactions (qpcr). The UDG in the master mix eliminates at least 10 5 copies of dutp-containing contaminating template. The SYBR Green I dye detects any double-stranded DNA that accumulates during the amplification process. The hot start feature increases sensitivity and specificity of SYBR -based qpcr by reducing the formation of primer-dimers and non-specific products. Simply add DNA template, primers, and water and the reactions are ready for cycling. Separate tubes of the passive reference dyes, ROX TM and fluorescein, are included for added convenience. High sensitivity and consistency over a broad dynamic range Detects fewer than 10 target copies Performs over a linear dynamic range of 7 to 8 orders of magnitude with a minimal correlation coefficient 0.95 Heat-labile UDG enables prevention of carry-over contamination Room temperature reaction setup Robust master mix withstands repeated freezethaws with no detectable loss of efficacy HotStart-IT SYBR Green qpcr Master Mix with UDG (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $ HotStart-IT Probe qpcr Master Mix with UDG (2X) HotStart-IT Probe qpcr Master Mix is supplied as a 2X premixed formulation containing HotStart-IT Taq DNA Polymerase, heat-labile UDG, MgCl 2, and Ultrapure nucleotides with an optimized dutp to dttp ratio for use in real-time quantitative PCR reactions (qpcr) with fluorescent probes. The UDG in the master mix eliminates at least 10 5 copies of dutpcontaining contaminating template. Use of fluorescent probes designed to hybridize to targets of interest increase detection specificity relative to non-specific dsdna binding dyes such as SYBR Green I. The Taq DNA Polymerase used in the master mix has the 5' 3' exonuclease activity necessary for efficient removal of the 5'-fluorophore from the 3'-quencher in TaqMan probes. Simply add DNA template, primers, probe(s) and water and the reactions are ready for cycling. A separate tube of ROX TM Passive Reference Dye is included for added convenience. HotStart-IT Probe qpcr Master Mix with UDG (2X) reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $659.00

8 qrt-pcr HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit provides optimal performance and maximum convenience for real-time, quantitative analysis of RNA templates in a single reaction format. HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit includes M-MLV RT, RNase Inhibitor, and a 2X Master Mix containing HotStart-IT Taq DNA Polymerase, MgCl 2, Ultrapure nucleotides, and SYBR Green I in an optimized reaction buffer. HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit Requires as little as 1 pg of total RNA or 100 fg of mrna High sensitivity and consistency over a broad dynamic range Detects fewer than 10 target copies Performs over a linear dynamic range of 6 orders of magnitude with a minimal correlation coefficient 0.95 Enables reverse transcription and qpcr in the same tube HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $1, R 2 = GAPDH Assay using HotStart-IT SYBR Green One-Step qrt-pcr Master Mix Kit (PN 75770). Duplicate reactions were performed with human placental total RNA as template using an ABI 7500 Fast instrument. The amount of template ranged from 100 ng to 1 pg in an order of magnitude dilution series. The nonspecific dsdna binding dye, SYBR Green I, was used to detect the 122 bp amplicon and ROX TM was used as a passive reference dye. The amplification process was linear over six orders of magnitude with a correlation coefficient of The No Template Control (NTC) reaction generated no measurable fluorescence. HotStart-IT Probe One-Step qrt-pcr Master Mix Kit HotStart-IT Probe One-Step qrt-pcr Master Mix Kit provides optimal performance and maximum convenience for real-time, quantitative analysis of RNA templates in a single reaction format. This Master Mix is a 2X pre-mixed formulation containing HotStart-IT Taq DNA Polymerase, MgCl 2, and Ultrapure nucleotides in an optimized reaction buffer for use with fluorescent probes in real-time, quantitative PCR reactions. Requires as little as 1 pg of total RNA or 100 fg of mrna High sensitivity and consistency over a broad dynamic range Detects fewer than 10 target copies Performs over a linear dynamic range of 6 to 7 orders of magnitude with a minimal correlation coefficient 0.95 Enables reverse transcription and qpcr in the same tube HotStart-IT Probe One-Step qrt-pcr Master Mix Kit reactions (50 µl rctn vol) $ reactions (50 µl rctn vol) $1, R 2 = GAPDH Assay using HotStart-IT Probe One-Step qrt-pcr Master Mix Kit (PN 75772). Duplicate reactions were performed with human placental total RNA as template using an ABI 7500 Fast instrument. The amount of template ranged from 1 µg to 10 pg in an order of magnitude dilution series. A TaqMan probe with FAM as the reporter fluorophore and BHQ-1 as the quencher was used to detect the 122 bp amplicon. ROX TM was used as a passive reference dye. The amplification process was linear over six orders of magnitude with a correlation coefficient of The No Template Control (NTC) reaction generated no measurable fluorescence.

9 First-Strand cdna Synthesis Kit for Real-Time PCR The First-Strand cdna Synthesis Kit for Real-Time PCR is optimized for reverse transcription of RNAs and produces first-strand cdna template suitable for realtime PCR. This kit includes an optimized Primer Mix which results in the generation of first-strand cdnas from an entire transcript without the end-bias observed with typical oligo(dt) n or random hexamer primers. This mixed primer strategy overcomes variability in real-time PCR gene expression analysis that can result from using different individual primers. For convenience, anchored oligo(dt) 23 VN and random hexamer primers are also included. Robust Performance: Optimized to reliably generate cdnas from full-length transcripts longer than 12 kb to be used in real-time PCR reactions. Sensitive: Mixed priming strategy overcomes 5' and/or 3' end bias associated with typical oligo(dt) n or random hexamer priming strategies, allowing sensitive and reliable real-time RT-PCR analysis of multiple user-defined targets from as little as 10 pg of starting total RNA. Consistent Performance: Optimized for generating first-strand cdna to be used in real-time PCR. HeLa total RNA and primers for qpcr are included as a positive control. Versatile: Three different priming strategies are provided to meet experimental needs. Gene-specific primers (GSP) may also be used (not included). First-Strand cdna Synthesis Kit for Real-Time PCR reactions (20 µl volume) $ The Primer Mix solution greatly reduces bias for sequences near the 5' and/or 3' ends of cdnas produced. The β-glucuronidase (GUSB) mrna was reverse transcribed from 100 ng of HeLa cell total RNA using (A) Random Hexamers, (B) Anchored Oligo(dT) 23 VN, or (C) the Primer Mix. Amplicons located near the 5' end (red) or 3' end (green) of the GUSB transcript were amplified by real-time PCR (on an ABI 7500 Fast instrument) using 1 μl of each reverse transcription reaction in 20 μl real-time PCR reactions (in duplicate) and HotStart-IT SYBR Green qpcr Master Mix (PN 75762). R 2 = Assay Flexibility and Consistent Priming Strategies. The First-Strand cdna Synthesis Kit for Real-Time PCR has been optimized for assay flexibility in carrying out reverse transcription using various priming strategies and diverse RNA templates. (A) Clathrin (NM_004859) and (B) Utrophin (NM_007124) amplicons were amplified using gene specific primers designed for real-time PCR (brown arrows). Reverse transcription was performed using HeLa cell total RNA (100 ng) and three different priming strategies: Anchored Oligo dt 23 VN primer (red), Random Hexamers (blue), and Primer Mix (green). 1 μl of each reverse transcription reaction was then used in 20 μl real-time PCR reactions on an ABI 7500 Fast instrument using HotStart-IT SYBR Green qpcr Master Mix (PN 75762). Assay Sensitivity and Dynamic Range. Various amounts of HeLa cell total RNA, ranging from 850 ng down to 10 pg, were reverse transcribed using anchored oligo(dt) 23 VN as the primer. 1 µl of each reverse transcription reaction was then used in 20 μl real-time PCR reactions (in duplicate) to amplify a 122 bp GAPDH amplicon (on an ABI 7500 Fast instrument), using HotStart-IT SYBR Green qpcr Master Mix (PN 75762). GAPDH amplification and linear correlation curve above show the wide dynamic range and sensitivity of the First-Strand cdna Synthesis Kit for Real-Time PCR. USB, the logo design, and HotStart-IT are registered trademarks of USB Corporation. FideliTaq and Tested User Friendly are trademarks of USB Corporation. HotStart-IT Taq DNA Polymerase Patent pending. Taq DNA Polymerase sold under licensing arrangements with Applied Biosystems. Purchase is accompanied by a limited license to use it in the Polymerase Chain Reaction (PCR) process in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by the up-front license fee, either by payment to Perkin-Elmer or as purchased, i.e., an authorized thermal cycler. The Polymerase Chain Reaction (PCR) is covered by patents owned by Roche Molecular Systems and F. Hoffmann-La Roche Ltd. UDG Purchase of this product is accompanied by a limited license under U.S. Patent Nos. 5,035,996; 5,683,896; 5,945,313; 6,518,026 and 6,287,823 and corresponding foreign patents. TaqMan is a registered trademark of Roche Molecular Systems, Inc. SYBR is a registered trademark of Molecular Probes, Inc. and is provided under an agreement with Molecular Probes, Inc. ROX is a trademark of Applera Corporation or its subsidiaries in the U.S. and certain other countries. BHQ-1 is a registered trademark of Biosearch Technologies Affymetrix, Inc. All rights reserved.

10 Power Your Research with Reliable USB PCR Products PCR Tools Nucleotides Molecular Biology Enzymes Molecular Biology Products & Kits USB, now part of Affymetrix, is an international leader in life sciences, from basic research to emerging technologies. We remain the partner you can trust for all your research needs. Our ISO 9001:2000 Certification assures our ongoing companywide commitment to quality, from product development to customer service. Purification RNA Analysis DNA Sequencing Biochemicals Affymetrix, Inc Miles Road Cleveland Ohio Customer Service: Phone: Fax: Technical Support: Phone: Fax: