Application Notes

Transcription

1 Application Notes Superior Performance and Flexibility In order to minimize errors in DNA replication during PCR, it is essential to choose a high-fidelity DNA polymerase enzyme. The introduction of errors using a standard DNA polymerase, such as (Thermus aquaticus DNA polymerase), can result in costly mistakes which can be difficult to identify in downstream cloning, site-directed mutagenesis and expression assays. DNA Polymerase offers both industry-leading fidelity and robust performance giving you results you can rely on with minimal optimization. DNA Polymerase is a single high performance proofreading polymerase offering extremely high fidelity and processivity. Consequently, PCR reaction times can be reduced significantly in comparison to other commercially available DNA polymerases. The unique properties of, combined with an optimized buffer system, allows for superior results with problematic templates such as high GC or AT content. Typical extension times for can be up to 45 seconds per kilobase and for proofreading polymerases, such as (Pyrococcus furiosus DNA polymerase), up to 2 minutes per kilobase. DNA Polymerase has been developed to significantly increase processivity, resulting in reduced extension times (10s/kb), thus reducing PCR turnaround time. In addition to providing high yield and robust performance, DNA Polymerase has the ability to amplify long templates in a fraction of the time of other commercially available proofreading DNA polymerases. Details of the superior performance and flexibility of DNA Polymerase are outlined in this application note. GC-rich templates PCR-amplification of GC-rich templates is often hampered by the formation of secondary structures, like hairpins and high melting temperatures, causing DNA polymerases to stall. This can result in low yields of the target fragment, ladders of nonspecific fragments, amplicons of the incorrect length, primerdimers and/or complete reaction failure. Routine amplification of GC-rich templates with commonly used proofreading DNA polymerases still remains unreliable. The unique properties of, combined with an optimized buffer system allows superior results, even when amplifying problematic templates. To demonstrate the success of DNA Polymerase in GC-rich PCR, was compared to two commonly used proofreading DNA polymerases, competitor P and. Amplification of different sized fragments of human genomic DNA was carried out with a GC content ranging from 66.9% to 76.9% (Fig. 1). The successful amplification of complex genomic fragments illustrates the robust performance of in comparison to competitor P and. Competitor P M M M M Fig. 1. Amplification of different types of GC-rich DNA fragments from human genomic DNA., a competitor DNA polymerase (P) and wild type were compared. Lanes 1 4 are a 728bp fragment of the GP150 gene (76.9% GC), a 724bp fragment of the MRGRE gene (68% GC), a 723bp fragment of the NM_ gene (66.9% GC) and a 788bp fragment of the NM_ gene (70.9% GC) respectively. Reactions were set up in 50μl using 25ng human genomic DNA and 0.2μM of each primer, 0.5μl dntps, 5% DMSO and the recommended PCR buffer and incubated at 95 C for 5min, followed by 30 cycles at 95 C for 30s, 60 C for 30s, and 72 C for 40s. 5μl was then run on a 1.5% TAE agarose gel. HyperLadder IV (M) (Cat No. BIO-33029). Yield The extreme efficiency of dramatically improves yields of PCR products while using fewer units of enzyme. One unit of was compared to 5 and 4 units of and polymerases respectively. A 400bp region of the human b-globin gene was amplified with decreasing numbers of cycles to demonstrate the improved yields seen with (Fig. 2). Not only are cycling times faster with, but PCR products can be obtained from fewer cycles than both and polymerases. M M M Fig. 2. Amplification of yield of PCR products with different polymerases. 100ng human genomic DNA was amplified to produce a 400bp fragment of human b-globin gene. Panel A. The reactions* were set up with 1 unit of DNA Polymerase. Reactions were incubated at 96 C for 2 min, and X cycles were performed at 98 C for 10s, 58 C for 5s, and 72 C for 20s, with a final 1min incubation at 72 C. Panel B and C. Reactions were set up with 2.5 units of DNA polymerase and 4 unit of Polymerase respectively. The reactions were incubated at 95 C for 2min, and X cycles were performed 95 C for 30s, 55 C for 30s, and 72 C for 30s. Where X is 30, 25, 20, 18 and 15 cycles (Lanes 1-5 respectively). HyperLadder I (M) (Cat No. BIO-33025). Application Notes

2 Fidelity The fidelity of a DNA polymerase is largely determined by the presence of an intrinsic 3-5 exonuclease (proofreading) activity, which can remove misincorporated bases The importance of proofreading is evident in comparisons of base substitution error rates between non-proofreading (10-4 to >10-5 ) and proofreading (10-6 to 10-7 ) DNA polymerases (Kunkel, T.A. 1992, Cline, J., et al. 1996). DNA Polymerase has been designed to increase speed without compromising fidelity. To demonstrate this, a modified laci-based fidelity assay was performed using a 1.9 kb sequence encoding lacioza. After amplification and cloning, the percentage of clones containing a mutation in laci (% blue) was determined in a color-screening assay (Provost G.S., et al. 1993), giving the error rate. was compared to,, Kod (Thermococcus kodakaraensis DNA polymerase) and VENT (Thermococcus litoralis DNA polymerase). Error Rate Kod VENT Polymerase Fig. 3. Error rates of different DNA polymerases determined in a color-screening using a modified laci-based fidelity assay. has been engineered to enhance fidelity so that it has industry-leading low error rates. The error rate of is as low as 4.4 x 10-7 (Fig. 3) which is 50-fold lower than that of, 7-fold better than and Kod and 5-fold lower than Vent DNA polymerase. Speed To test the speed of, a 1.3kb fragment from the human angiotensin receptor type-1 gene was amplified using, and DNA Polymerase across a range of decreasing extension times. Fig. 4. shows that requires shorter extension times than proofreading polymerases such as, however due to its high processivity, has significantly shorter extension times than both and (Fig. 4). Amplification of a 1.3 kb fragment from the human angiotensin receptor type-1 gene from human genomic DNA, was possible using with extension times as short as 10s/kb. Furthermore, the unique property of means that denaturation can be carried out at temperatures as high as 98 C, reducing the time required for this step. delivers faster cycling times and PCR results quicker than ever (see Fig. 5). M M Fig. 4. Amplification of the human angiotensin receptor gene. 100ng human genomic DNA (Cat No. BIO-35025) was amplified with primers to produce a 1.3-kb fragment of human angiotensin receptor type-1 gene. Panel A. 1 unit of DNA Polymerase was used per reaction. The reactions* were incubated at 96 C for 2min, and 25 cycles were performed at 98 C for 10s, 58 C for 5s, and 72 C for 60, 45, 30, 20 or 10s (Lanes 1-5 respectively). Panel B and C. Reactions were set up with 2.5 units of and 4 units of respectively. The reactions were incubated at 95 C for 2 min, and 25 cycles were performed at 95 C for 2mins, 55 C for 30s, and 72 C for 60, 45, 30, 20 or 10s (Lanes 1-5 respectively). HyperLadder I (M) (Cat No. BIO-33025). Size (Kb) Time (min) Fig. 5. Estimated PCR extension times for different DNA Polymerases based around standard protocols and 25x cycles. Reduced denaturation and extension steps for DNA Polymerase result in shorter PCR runs and improved turnaround times. Application Notes

3 Application Notes Processivity Processivity is defined as the number of nucleotides incorporated by the polymerase per binding event and is determined by extension rate and DNA-binding affinity. To examine the processivity of DNA Polymerase, fragments of human genomic DNA up to 9kb were amplified. The results (Fig. 6) confirm that can successfully amplify fragments of up to 9kb from genomic DNA without significant loss of yield due to its high processivity. ROBUST Proofreading DNA polymerases tend to require optimization, especially in the case of the amplification of difficult templates containing high GC or AT content. provides robust amplification without compromising fidelity., and were used to amplify a 750bp fragment of the human p64 gene (with 65% GC content) from both cdna and genomic DNA. The amplification of complex genomic fragments such as the human p64 gene illustrates the robust performance of in comparison to and. requires minimal PCR optimization as it is capable of amplifying difficult templates. M M Fig. 6. Processivity of DNA Polymerase. 100ng genomic DNA was amplified to produce 9.0, , 1.5 and 0.6kb fragments of human genomic DNA (Lanes 1-5 respectively). Reactions* were set up with 1 unit of DNA Polymerase and incubated at 96 C for 5min, and 35 cycles were performed at 96 C for 10s, 55 C for 1min, and 72 C for 9min. HyperLadder I (M) (Cat No. BIO-33025). (*50μl reactions were assembled on ice according to the manufacturers instructions. After amplification 10μl of the PCR products was run out on a 1.0% TAE agarose gel (Cat No. BIO-41026) and stained with 0.25 μg/ml ethidium bromide.) is also capable of processing templates in the presence of inhibitors such as debris fom cell suspensions (Fig. 7). Sensitivity Advances in PCR have led to a demand for amplification from increasingly small quantities of template DNA. This can prove to be a major limitation for some proofreading DNA polymerases owing to the relatively poor sensitivity of these enzymes. To demonstrate the superior sensitivity of, a serial dilution of genomic DNA was used to amplify a 2.4kb fragment of the human a-1-antitrypsin gene with, and. The results highlight that even with difficult templates (Fig. 8), DNA Polymerase provides superior results at low template concentrations. M M Fig. 7.Robust performance of DNA Polymerase. 750bp fragment of the human p64 gene. (Lanes ng, 10ng and 1ng cdna and 100ng genomic DNA respectively. Reactions* were performed using DNA Polymerse and incubated at 96 C for 5min, and 25 cycles at 98 C for 10sec, 58 C for 15sec, and 72 C for 30secs (Lanes 5-6) using 100ng genomic DNA and 100ng cdna respectively. 2.5 units of DNA Polymerase was used and incubated at 95 C for 5min, and 25 cycles were performed at 95 C for 30sec, 55 C for 30sec, and 72 C for 42secs (Lanes 7-8) using 100ng genomic DNA and 100ng cdna respectively. 4 units of DNA Polymerase was used and incubated at 95 C for 5min, and 25 cycles were performed at 95 C for 30sec, 55 C for 30sec, and 72 C for 60secs. In all reactions a final 1min incubation at 72 C was performed. Marker is HyperLadder I (Cat No. BIO-33025). A) B) C) M M M Fig. 8. DNA polymerase sensitivity was tested by PCR using decreasing concentrations of template DNA. A 2.4kb fragment of the human a-1-antitrypsin gene was amplified using 100, 50, 25, 12.5, 6.25, 3.125, 1.5, 0.75, 0.35 and 0.17ng of DNA (lanes 1-10 respectively). Panel A. Reactions were set up with DNA Polymerase and incubated at 96 C for 5 min, and 25 cycles were performed at 98 C for 10s, 58 C for 15s, and 72 C for 2min. Panel B. Reactions were set up with 2.5 units of DNA polymerase and incubated at 95 C for 5 min, and 25 cycles were performed at 95 C for 30s, 55 C for 30s, and 72 C for 2min 30s. Panel C. Reactions were set up with 4 units of DNA Polymerase and incubated at 95 C for 5 min, and 25 cycles were performed at 95 C for 30s, 55 C for 30s, and 72 C for 3min. HyperLadder I (M) (Cat No. BIO-33025).

4 Application Notes Summary DNA Polymerase is a novel, single-enzyme that exhibits industry-leading performance in terms of speed, processivity and fidelity. not only exhibits extremely high-fidelity, out-performing other proofreading enzymes, but also offers superb yield, processivity and speed normally only associated with non-proofreading polymerases. This combination of factors makes suitable for all PCR applications which require great accuracy such as site-directed mutagenesis or cloning, and also making it a superior choice for difficult templates (such as GC-rich). The reliability of allows users to reduce extension times and overall cycling times when compared to other commercialy available proofreading polymerases. Please visit to request a sample of. Bioline Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel: +44 (0) Fax: +44 (0) USA Bioline USA Inc. 305 Constitution Dr. Taunton, MA Toll Free: Tel: Fax: Germany Bioline GmbH Im Biotechnologiepark TGZ 2 D Luckenwalde Tel: +49 (0) Fax: +49 (0) Bioline (Aust) Pty Ltd PO Box 122 Alexandria NSW 1435 Tel: +61 (0) Fax: +61 (0) Notes: 1. and HyperLadder are trademarks of Bioline. AN0511V1.1

5 Application Notes DNA Polymerase Advantages of using DMSO Templates with GC-rich or long complementary areas can be difficult to amplify, as they possess stable secondary structures that resist denaturation and prevent primer annealing. There are several PCR additives, such as DMSO, that make denaturation easier by relaxing the template DNA. We have found however, that the addition of dimethyl sulfoxide (DMSO) to any reaction using DNA Polymerase, regardless of amplicon length or composition, increases amplification efficiency and specificity. Bioline has developed DNA Polymerase, an ultra-fast thermostable enzyme possessing 3-5 proofreading exonuclease activity. delivers outstanding PCR yield with exceptional fidelity, even from low template concentrations (fig. 1). encompasses the best of all polymerase functionality in one enzyme, making it the perfect choice for most PCR applications. Supplier P M Fig. 1. Amplification of human genomic DNA A/ a 1kb and B/ a 10kb fragment was amplified from 10ng, 2ng, 400pg, 80ng, 16pg, 3.2pg, 0.6pg and 0.1pg (lanes 1-8 respectively) of human genomic DNA using and supplier P. Reactions were incubated at for 98 C for 2 min followed by 30 cycles at 98 C for 30s, 55 C, for 30s, and 72 C for 1 or 10 min. HyperLadder I (M). PCR amplification of GC-rich templates is often hampered however, by the formation of secondary structures like hairpins and higher melting temperatures, causing DNA polymerases to stall. This can result in low yields, ladders of non-specific fragments, amplicons of the incorrect length, primer-dimers and/or complete reaction failure. Routine amplification of GC-rich templates with commonly used high-fidelity DNA polymerases therefore still remains unpredictable. A variety of additives can be included in PCR amplifications to increase yield, specificity and consistency, one of the most common being DMSO (fig. 2). Supplier P M M M M Fig. 2. Amplification of GC-rich DNA fragments from human genomic DNA, Supplier P polymerase and wild-type were compared. Lanes 1 4 correspond to the amplification of a 728bp fragment of the GP150 gene (76.9% GC), a 724bp fragment of the MRGRE gene (68% GC), a 723bp fragment of the NM_ gene (66.9% GC) and a 788bp fragment of the NM_ gene (70.9% GC) respectively. Reactions were set up in 50µl using 25ng human genomic DNA and 0.2µM of each primer, 1mM dntps, 5% DMSO and the recommended PCR buffer and incubated at 98 C for 5 min, and 25 cycles of 98 C for 30s, 55 C for 30s, and 72 C for 45s. HyperLadder IV (M). We have found that DMSO not only enhances long and GC-rich DNA, but also improves yields and sensitivity with less complex and shorter DNA (fig. 3). No DMSO Supplier P No DMSO 3% DMSO Supplier P 3% DMSO A/ B/ C/ Fig. 3. Benefits of using DMSO with A serial dilution of human genomic DNA - 100ng, 33ng, 10ng, 3.3ng, 1ng and 330pg (lanes 1-6 respectively) was amplified using three sets of primers of increasing amplicon length (890bp, 2598bp and 3915bp respectively). Reactions were set up in 50µl using 25ng human genomic DNA and 0.2µM of each primer, 1mM dntps, 3% DMSO and the recommended PCR buffer and incubated at 98 C for 2 min, and 30 cycles of 98 C for 30s, 55 C for 30s, and 72 C for A/ 1 min, B/ 3 min and C/ 4 min. HyperLadder I (M). Application Notes

6 Application Notes Summary The 5x Hi-Fi Buffer and has been designed to give high yield and fidelity for the majority of standard templates; however based on the results above we would therefore recommend the addition of 3% DMSO (final concentration) for optimal performance of all PCR reactions. For more difficult templates possessing even higher GC-content or complex structural organization, we would recommend doing a titration up to 10% DMSO, however in this case the annealing temperature should be reduced since DMSO decreases the melting temperature of primers by up to 5 C. Please visit to request a sample of. Bioline Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel: +44 (0) Fax: +44 (0) USA Bioline USA Inc. 305 Constitution Dr. Taunton, MA Toll Free: Tel: Fax: Germany Bioline GmbH Im Biotechnologiepark TGZ 2 D Luckenwalde Tel: +49 (0) Fax: +49 (0) Bioline (Aust) Pty Ltd PO Box 122 Alexandria NSW 1435 Tel: +61 (0) Fax: +61 (0) Note: HyperLadder is a trademark of Bioline. AN0211V1.2