Navigation Links
Choice of RT-PCR Enzymes

Obviously, in RT-PCR a major factor to consider is the choice of reverse transcriptase used to synthesize cDNA. Since each of the available enzymes has different enzymatic properties, one may be more suitable for a specific experiment than the others. Among the enzyme properties to consider are: Temperature optima

Higher incubation temperatures can help eliminate problems of template secondary structure. In addition, high temperature improves the specificity of reverse transcription by decreasing false priming. Thus, thermoactive reverse transcriptases that can be incubated at high temperatures (5070C) are more likely to produce accurate copies of mRNA, especially if the template has a high GC content.

Note: At these high temperatures, use only specific primers; do not use oligo(dT) or random hexamer primers.

RNase H activity

In contrast to Tth DNA Polymerase, AMV and M-MuLV Reverse Transcriptases have RNase H activity. This activity will specifically degrade RNA in an RNA:cDNA hybrid. This can be detrimental if degradation of template RNA competes with DNA synthesis during production of first-strand cDNA. M-MuLV Reverse Transcriptase has lower RNase H activity than AMV Reverse Transcriptase. Mutated enzymes without RNase H activity are also available, e.g. Expand Reverse Transcriptase (Cat. No. 1785826) or the C. therm. Polymerase for Reverse Transcription in Two-Step PCR (Cat. No. 2016311) from Roche Applied Science.

Note: Expand Reverse Transcriptase is not for sale in the United States.

Divalent ion requirement Most reverse transcriptases require a divalent ion for activity. Enzymes that use Mg2+ are likely to produce more accurate cDNA copies than those that use Mn2+, since Mn2+ adversely affects the fidelity of DNA synthesis. Specificity and sensitivity Reverse transcriptases have differing ability to copy small amounts of template (sensitivity). They also differ in their ability to transcribe RNA secondary structures accurately (specificity). Reverse transcriptase DNA polymerase combinations

Using different reverse transcriptase DNA polymerase combinations can allow you to optimally amplify certain length targets.

The properties of reverse transcriptases and their suggested applications are given in the tables listed below.

Properties of reverse transcriptases Suggested applications for Reverse Transcriptases Reverse transcriptases suitable for one-step RT PCR Reverse transcriptases suitable for two-step RT PCR
'"/>

Source:


Page: All 1 2 3

Related biology technology :

1. TripleMaster PCR SystemThe Ideal Choice for Long-Range PCR
2. Choice of reverse transcription primers
3. Microdetermination of Phosphorus Using the SpectraMax Plus Microplate Spectrophotometer: Choice of Microplate, Cuvette, or Test Tube Assay Formats (MaxLine Application Note #24)
4. New Kit Generates Exceptionally Pure Total RNA for RT-PCR
5. Brilliant Core Reagent Kits for QPCR and QRT-PCR
6. Control RT-PCR Primers for Human Gene Transcripts with Varying Abundance
7. prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification
8. Performance Comparisons of Commercial RT-PCR Systems
9. RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes
10. RT-PCR Primers for the Study of Apoptosis
11. cMaster RTplusPCR System for increased sensitivity and product length in RT-PCR
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:1/24/2017)... ... January 24, 2017 , ... ZeptoMetrix Corporation and ... panels with standard concentrations of histidine rich protein 2 (HRP2). , The ... concentrations from six different malaria strains, are intended for use by researchers and ...
(Date:1/24/2017)... ... January 23, 2017 , ... The ... through unique partnerships, seeks outstanding early career nominees for the 2017 New Innovator ... meeting the needs of a world in which one in nine people suffers ...
(Date:1/24/2017)... CORAL GABLES, Fla. , Jan. 23, 2017 ... interdisciplinary network focused on scientific discovery and solutions, ... is creating the Frost Institutes for Science and ... technology, engineering, and mathematics (STEM) to help solve ... University of Miami President Julio Frenk ...
(Date:1/24/2017)... , Jan. 23, 2017  Today, the Fisher ... they have funded an important study that could lead ... the onset of Alzheimer,s disease. This groundbreaking research was ... University, led by Nobel Laureate Dr. Paul Greengard ... Alzheimer,s disease. Fisher Center scientists have linked ...
Breaking Biology Technology:
(Date:12/16/2016)...   IdentyTechSolutions America LLC , a leading ... and a cutting-edge manufacturer of software and hardware ... seamless, integrated solutions that comprise IDT biometric readers ... provide IdentyTech,s customers with combined physical identification and ... and theft. "We are proud to ...
(Date:12/16/2016)... NEW YORK , Dec. 16, 2016 The global ... reach USD 12.14 billion by 2021 from USD 5.31 billion in ... ... market is mainly driven by technological advancements in medical devices, launch ... devices, rising preference for wireless connectivity among healthcare providers, and increasing ...
(Date:12/15/2016)... 15, 2016   WaferGen Bio-systems, Inc. (NASDAQ: ... company, announced today that on December 13, 2016, it ... The Nasdaq Stock Market LLC which acknowledged that, as ... WaferGen,s common stock had been at $1.00 or greater ... compliance with Listing Rule 5550(a)(2) of the Nasdaq Stock ...
Breaking Biology News(10 mins):