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:5/23/2016)... ... May 23, 2016 , ... PrecisionAg® ... Farming in 2017 and Beyond. The paper outlines the key trends that are ... industry. , “We’ve witnessed a lot of highs and lows as the precision ...
(Date:5/23/2016)... CA (PRWEB) , ... May 23, 2016 , ... ... public interest organization focused on molecular nanotechnology, announced the winners for the 2015 ... pioneer physicist Richard Feynman, are given in two categories, one for experiment and ...
(Date:5/20/2016)... Baltimore, MD (PRWEB) , ... May 20, 2016 , ... ... contaminated with Listeria, as reported by Food Safety News on May 12, 2016(1), demonstrates ... according to Ted Olsen, CEO of Baltimore-based biotech firm, PathSensors, Inc. ...
(Date:5/19/2016)... May 19, 2016 ... (OTC PINK: RGBPP) announced today initiation of a ... cord blood based cancer immunotherapeutic product leveraging its ... Regen described a generation of cord blood derived ... gene silencing.  The product in development will be ...
Breaking Biology Technology:
(Date:3/14/2016)... http://www.apimages.com ) - ... - Renvoi : image disponible via AP Images ( ... --> DERMALOG, le leader de l,innovation ... d,empreintes digitales pour l,enregistrement des réfugiés en Allemagne. ... produire des cartes d,identité aux réfugiés. DERMALOG dévoilera ...
(Date:3/10/2016)... March 10, 2016   Unisys Corporation (NYSE: ... Protection (CBP) is testing its biometric identity solution at ... to help identify certain non-U.S. citizens leaving the ... test, designed to help determine the efficiency and accuracy of ... February and will run until May 2016. --> ...
(Date:3/8/2016)... 2016   Valencell , the leading innovator ... has secured $11M in Series D financing. The ... venture fund being launched by UAE-based financial services ... investors TDF Ventures and WSJ Joshua Fund. Valencell ... triple-digit growth and accelerate its pioneering innovation in ...
Breaking Biology News(10 mins):