Jun 15, 2020
One enzyme reverse transcription qPCR using Taq DNA polymerase
This protocol is a draft, published without a DOI.
- Sanchita Bhadra1,
- Andre Maranhao1,
- Andrew Ellington1
- 1The University of Texas at Austin
- Reclone.org (The Reagent Collaboration Network)Tech. support email: protocols@recode.org
External link: https://doi.org/10.1101/2020.05.27.120238
Protocol Citation: Sanchita Bhadra, Andre Maranhao, Andrew Ellington 2020. One enzyme reverse transcription qPCR using Taq DNA polymerase. protocols.io https://protocols.io/view/one-enzyme-reverse-transcription-qpcr-using-taq-dn-bhicj4aw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 14, 2020
Last Modified: June 15, 2020
Protocol Integer ID: 38180
Keywords: Taq, RT-qPCR, One enzyme RT-qPCR, SARS-CoV-2,
Abstract
Taq DNA polymerase, one of the first thermostable DNA polymerases to be discovered, has been typecast as a DNA-dependent DNA polymerase commonly employed for PCR. However, Taq polymerase belongs to the same DNA polymerase superfamily as the Molony murine leukemia virus reverse transcriptase and has in the past been shown to possess reverse transcriptase activity. We report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase, and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions. We demonstrate the utility of Taq-alone RT-qPCR reactions by executing CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays that could detect as few as 2 copies/μL of input viral genomic RNA.
Attachments
Materials
Chemicals and reagents
All chemicals were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise indicated. All enzymes and related buffers were purchased from New England Biolabs (NEB, Ipswich, MA, USA), Thermo Fisher Scientific (Waltham, MA, USA), or Promega (Madison, WI, USA) unless otherwise indicated. All oligonucleotides and TaqMan probes (Table 1) were obtained from Integrated DNA Technologies (IDT, Coralville, IA, USA). SARS-CoV-2 N gene armored RNA was obtained from Asuragen (Austin, TX, USA). SARS-CoV2 viral genomic RNA was obtained from American Type Culture Collection (Manassas, VA, USA).
Preparing RT-qPCR reaction
Preparing RT-qPCR reaction
30m
30m
RT-qPCR assays were assembled in a total volume of 25 µL containing the indicated buffer at 1X strength (see Generation 6A buffer recipe in step 2)
Optimized buffer recipe
Optimized buffer recipe
In this study, we report optimized buffer and salt compositions that promote the reverse transcriptase activity of Taq DNA polymerase and thereby allow it to be used as the sole enzyme in TaqMan RT-qPCR reactions.
CITATION
We recommend to use Generation 6A buffer for the one enzyme RT-qPCR.
10X Generation 6A buffer (600 millimolar (mM) Tris-HCl ,8.0 , 20 millimolar (mM) (NH₄)₂SO₄ , 400 millimolar (mM) KCl , 20 millimolar (mM) MgCl2 )
Preparing RT-qPCR reaction
Preparing RT-qPCR reaction
30m
30m
The buffer was supplemented with 0.4 millimolar (mM) deoxyribonucleotides (dNTP) , 402 nanomolar (nM) each of forward and reverse PCR primer pairs , 102 nanomolar (nM) of the TaqMan probe , and 2.5 units of Taq DNA polymerase from indicated commercial vendors.
15m
Indicated copies of SARS-CoV-2 viral genomic RNA, SARS-CoV-2 N gene armored RNA, or RNaseP armored RNA prepared in TE buffer (10 millimolar (mM) Tris-HCl , 7.5 , 0.1 millimolar (mM) EDTA , 8.0 ) immediately prior to use were added to RT-qPCR reactions containing corresponding PCR primers. Negative control reactions did not receive any specific templates.
15m
Running RT-qPCR
Running RT-qPCR
2h
2h
Amplicon accumulation was measured in real-time by incubating the reactions in a LightCycler96 qPCR machine (Roche, Basel, Switzerland) programmed to hold 60 °C for 00:30:00 followed by 95 °C for 00:10:00 prior to undergoing 55 cycles of 95 °C for 00:00:15 and 60 °C for 00:00:30 . TaqMan probe fluorescence was measured during the amplification step (60 °C for 30 sec) of each cycle using the FAM channel.
2h
Citations
Step 2
Sanchita Bhadra*, Andre C. Maranhao*, and Andrew D. Ellington. One enzyme reverse transcription qPCR using Taq DNA polymerase
https://doi.org/10.1101/2020.05.27.120238