Authors: Rachel Graham
This protocol details the primers and conditions used for forward and reverse PCR amplification and sequencing of SARS-CoV genomes.
This protocol details the steps, reagents, and conditions required to sequence SARS-CoV genomes in the forward and reverse directions. The protocol begins with the RT-PCR step, assuming that SARS-CoV RNA purified using a standard procedure (i.e., TRIzol extraction) has already been performed.
- Invitrogen SuperScript III kit
- dNTPS (10 mM)
- Random Hexamers
- RNasin (if desired)
- Thermo Phusion PCR enzyme
- Primers (2 mM stock – see Tables 1 and 2)
- 1X TAE Buffer
- Ethidium Bromide
- 70ºC water bath
- 55ºC water bath
- Thermal cycler
- In a 1.5-µL Eppendorf tube, add 1-4 µL RNA and 1 µL Random Hexamers.
- Incubate at 70ºC for 5 min.
- Place reaction briefly on ice and assemble the reverse transcription reaction using the kit-supplied reagents (a master mix can be made if multiple reactions are being run):
- 4 µL 5X First-Strand Buffer
- 2 µL DTT
- 1 µL SuperScript III reverse transcriptase
- 1 µL dNTPs
- 1 µL RNasin (if desired)
- x µL H2O to 20 µL
- Incubate at 55ºC for 45 min to 1 h.
- Inactivate the reverse transcriptase at 70ºC for 15 min. Place reaction on ice after inactivation.
- Proceed with PCR setup.
PCR (with Phusion PCR kit):
- Assemble PCR reactions to generate amplicons according to those detailed in Table 2 (for whole-genome sequencing) or with any combination of forward and reverse primers from Table 1.
- PCR reaction setup:
- 2 µL First-strand template
- 1 µL Forward Primer
- 1 µL Reverse Primer
- 5 µL 10X HF Buffer
- 1 µL dNTPs
- 0.5 µL Phusion polymerase
- x µL H2O to 50 µL
- PCR reactions are run under standard PCR conditions:
- 98ºC 5 min
- 35 cycles of:
- 98ºC 15 sec
- xºC for 30 sec*
- 72ºC for ~45 sec/kb
- 72ºC 10 min
- 8ºC Hold
Annealing temperature is primer-dependent, but for most SARS-CoV primers in Table 1, annealing temperatures 52-55ºC will work.
Confirmation of PCR products and sequencing:
- Run PCR products (5 µL/reaction) on a 0.8% agarose/1X TAE gel to verify PCR success.
- Purify PCR products with PCR purification kit of choice (the Qiagen PCR Purification Kit works well).
- PCR products can be diluted to 150-200 µL/reaction to ensure that enough product is present for assembling sequencing reactions.
- Assemble sequencing reactions according to the primer/amplicon combinations outlined in Table 2.
- Reverse transcription: 1-1.5 h
- PCR: 2-4 h
- Sequencing: facility-dependent
Primers have been designed to give clean, single-band PCR products. If multiple bands are detected, alternate annealing temperatures may be required. It may also be possible that alternate bands indicate multiple genome patterns at that locus.
*Table 1: SARS-CoV PCR/Sequencing Primers *
Table 2: SARS-CoV Amplicon Primer Sets
This protocol is related to the following articles:
A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease, Rachel L Graham, Michelle M Becker, Lance D Eckerle, Meagan Bolles, Mark R Denison, and Ralph S Baric, Nature Medicine 18 (12) 1820 - 1826
Source: Protocol Exchange. Originally published online 10 July 2014.