Navigation Links
Improve Amplification Specificity with Hot Start PCR Enzyme


SureStart Taq DNA polymerase utilizes ultra-pure Taq2000 DNA polymerase

SureStart Taq DNA polymerase features a modified version of Taq2000 DNA polymerase with hot start capability. Using SureStart Taq, hot start is in- corporated into PCR protocols previously optimized with Taq DNA polymerase, with little or no modification of cycling parameters or reaction conditions. SureStart Taq can be used in a variety of amplification systems to improve specificity, yield, and amplification of difficult targets.

A 105 bp fragment of the glucocerebrosidase gene was amplified from human genomic DNA using SureStart Taq (lane 1), unmodified Taq (lane 2), a competitors antibody-based hot start Taq (lane 3), or a competitors modified Taq (lane 4) DNA polymerase preparation. PCR reactions were assembled at room temperature and employed 2.5 U of each enzyme and the recommended buffer. Identical temperature cycling parameters were used, except that a heat step 95C, 10 minute heat step was added to the beginning of the thermal cycling program for lanes 1 and 4.

Combining PCR reactants at low temperatures can lead to high background and low product yields in some amplification systems. Certain PCR enzymes exhibit significant polymerase activity at temperatures encountered during reaction set-up (4 to 25C) or while ramping up to stringent primer annealing temperatures. For example, Taq DNA polymerase exhibits 2 to 3% maximum activity at 25C (room temperature PCR set up) and 70% maximum activity at 50C (generally at or below the Tm of PCR primers)1. Non-specific primer annea ling and extension at non-restrictive temperatures produces undesirable products that are amplified throughout the remaining PCR cycles. Misprimed products and artifacts such as primer-dimers can impair gel analysis, quantitation, and sequencing of specific PCR products. Moreover, in the amplification of misprimed products, dNTPs and primers are diverted away from the synthesis of specific products, reducing overall yields.

Stratagenes SureStart Taq DNA polymerase improves amplification reactions by reducing background and increasing yield of desired product.

SureStart Taq DNA polymerase consists of a reversibly inactivated version of Stratagenes Taq2000 DNA polymerase, a highly purified recombinant Taq DNA polymerase preparation. SureStart Taq polymerase remains inactive until stringent denaturation temperatures are reached. It is activated by adding a heat step (9 to 12 minutes at 92 to 95C) to the beginning of thermal cycling programs (pre-PCR heat-activation method). Alternatively, the pre-PCR heat-activation step can be omitted to permit enzyme activation during temperature cycling. When the PCR heat-activation method is employed, it is generally necessary to add additional cycles to existing cycling programs to achieve optimal product yield.

SureStart Taq DNA polymerase provides superior or equivalent improvements in specificity and product yield compared to other vendors hot start Taq preparations. In the amplification system shown in the figure, Taq DNA polymerase synthesizes multiple extraneous bands in addition to the desired 105 bp fragment (lane 2). In contrast, background is eliminated in reactions employing SureStart Taq DNA polymerase (lane 1). SureStart Taq DNA polymerase provided improved specificity and higher product yield compared to other vendors antibody-based (lane 3) and modified (lane 4) Taq hot start formulations. Moreover, using SureStart Taq DN A polymerase, superior performance and the convenience of room temperature PCR setup was easily achieved with only minor changes to the temperature cycling program.

REFERENCES

  1. Nielson, K.B., Cline, J., and Hogrefe, H. (1997) Strategies 10: 40-43.


'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. The Best Enzyme for the Toughest PCR Challenges Improved with New Hot-Start Feature
2. Novel PCR Enhancing Factor Improves Performance of Pfu DNA Polymerase
3. Improved Sensitivity for Staining RNA with SYBR Gold Stain
4. Improve Lipid- or Calcium Phosphate-Mediated Transfection of Human Dermal Fibroblasts
5. Improved Vectors for PathDetect Trans-Reporting Systems
6. Improved Quantitative Selectivity of Clenbuterol in Human Urine using High Resolution on the TSQ Quantum Mass Spectrometer
7. Gene Expression Arrays: Highly Sensitive Detection of Expression Patterns with Improved Tools for Target Amplification
8. Improve RNA Isolation for Array Analysis and More
9. Protect RNA and Improve Target Cell Identification
10. The Worlds Best DNase Improved TURBO DNA-free
11. Improve Recovery and Reproducibility with Pre-Treatment of Analytical TSK-GEL G3000SWXL Size Exclusion Columns
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:6/27/2016)... June 27, 2016   Ginkgo Bioworks , a ... engineering, was today awarded as one of the ... the world,s most innovative companies. Ginkgo Bioworks is ... the real world in the nutrition, health and ... directly with customers including Fortune 500 companies to ...
(Date:6/23/2016)... Seattle, WA (PRWEB) , ... June 23, 2016 ... ... technology, announces the release of its second eBook, “Clinical Trials Patient Recruitment and ... patient recruitment and retention in this eBook by providing practical tips, tools, and ...
(Date:6/23/2016)... 23, 2016 Houston Methodist Willowbrook Hospital ... Sports Association to serve as their official health ... Methodist Willowbrook will provide sponsorship support, athletic training ... association coaches, volunteers, athletes and families. ... Sports Association and to bring Houston Methodist quality ...
(Date:6/23/2016)... , ... June 23, 2016 , ... ... announced the launch of the Supplyframe Design Lab . Located in Pasadena, ... explore the future of how hardware projects are designed, built and brought to ...
Breaking Biology Technology:
(Date:3/31/2016)... 2016   LegacyXChange, ... "Company") LegacyXChange is excited to release its ... to be launched online site for trading 100% guaranteed ... will also provide potential shareholders a sense of the ... an industry that is notorious for fraud. The video ...
(Date:3/23/2016)... Massachusetts , March 23, 2016 /PRNewswire/ ... im Interesse erhöhter Sicherheit Gesichts- und Stimmerkennung ... Xura, Inc. (NASDAQ: MESG ), ... bekannt, dass das Unternehmen mit SpeechPro zusammenarbeitet, ... aus der Finanzdienstleistungsbranche, wird die Möglichkeit angeboten, ...
(Date:3/21/2016)... Massachusetts , March 22, 2016 ... facial recognition with passcodes for superior security   ... ), a leading provider of secure digital communications services, ... their biometric technology and offer enterprise customers, particularly those ... secure facial recognition and voice authentication within a mobile ...
Breaking Biology News(10 mins):