Thirty-two pairs of nested PCR assays that span the genome were designed based on alignment of available MERS-CoV genomes.39 (link), 40 (link) MERS-CoV genomes or partial genomes were amplified as needed to fill the gaps missed by Fluidigm Access Array PCR. Positive bands of the expected size that had strong signal and without additional bands were cleaned up using Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Roche). Samples were incubated at 37 °C for 15 min followed by 80 °C for 15 min to inactivate the Exonuclease and Shrimp Alkaline Phosphatase. Purified PCR amplicons were sequenced with the PCR primers in both directions on an ABI Prism 3130 Automated Capillary Sequencer (Applied Biosystems, Foster City, CA, USA) using Big Dye 3.1 cycle sequencing kits (Life Technologies, Carlsbad, CA, USA). The Sanger sequence data were analyzed using Sequencher 5.0. Ends and low-quality regions were trimmed manually. Contigs and consensus sequences were generated.
Big dye 3.1 cycle sequencing kits
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Big Dye 3.1 cycle sequencing kits from Thermo Fisher Scientific are a set of reagents used for DNA sequencing. The kits contain the necessary components, including fluorescently labeled dideoxy nucleotides, to perform the Sanger sequencing method.
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Lab products found in correlation
4 protocols using big dye 3.1 cycle sequencing kits
1
MERS-CoV Genome Sequencing Protocol
2
Sanger Sequencing of PCR Amplicons
3
Sanger Sequencing of PCR Amplicons
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Positive bands of the expected size that had strong signal and without additional bands were cleaned up using Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Roche). Samples were incubated at 37°C for 15 minutes followed by 80°C for 15 minutes to inactivate the Exonuclease and Shrimp Alkaline Phosphatase. Positive bands of the expected size with additional bands present in the PCR products were purified using QIAquick Gel Extraction kits (Qiagen). Purified PCR amplicons were sequenced with the PCR primers in both directions on an ABI Prism 3130 Automated Capillary Sequencer (Applied Biosystems) using Big Dye 3.1 cycle sequencing kits (Life Technologies).
4
Pan-viral Molecular Diagnostics Protocol
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Pan-viral family/genus PCRs and sequencing were performed for the following viral families/genera: Coronaviridae, Herpesviridae, Orthomyxoviridae (Influenza viruses A, B and C), and Reoviridae (Aquareovirus and Orthoreovirus) [33–36 (link)]. First round reverse transcription PCR for RNA viruses was performed with Superscript III/Platinum Taq One Step kits (Invitrogen) and Titanium Taq (Clontech) kits for the second round PCR. First and second round PCR for DNA viruses was performed with Hot Start Ex Taq kits (Takara). A positive PCR control containing mutation-engineered synthetic RNA transcript or DNA amplicon and a negative control using nuclease-free water were included in each run. PCR products were visualized on 2% agarose gels. Positive bands of the expected size that had a strong signal and without additional bands were purified using Exonuclease I (New England Biolabs) and Shrimp Alkaline Phosphatase (Roche). Samples were incubated at 37°C for 15 min followed by 80°C for 15 min to inactivate the Exonuclease and Shrimp Alkaline Phosphatase. Purified PCR amplicons were sequenced with the PCR primers in both directions as described previously [36 ] on an ABI Prism 3130 Automated Capillary Sequencer (Applied Biosystems) using Big Dye 3.1 cycle sequencing kits (Life Technologies).
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