C-terminal tag protocol: Adding a C-terminal tag to a gene

Peccoud Lab Protocol: designing primers to add a C-terminal tag to the (3′) end of a gene via PCR

Introduction

It is sometimes necessary to generate gene fusion strains with a C-terminal tag or promoter changes by intoducing a PCR amplicon of a selectable marker flanked by DNA sequences homologous to the the last 40-60 bp of the ORF before the STOP codon (protein fusions) or after the STOP codon (mRNA tags). This protocol describes the design process in making yeast strains carrying gene fusions at the 3′ end of ORFs (C-terminal tag).

Method

  1. Check LabCollector for Existing Stocks of the Desired Primers Before Starting the Design Process
  2. Plasmid Sequence:
    Decide which C-terminal tag and selectable marker combination you wish to fuse to the gene of interest (GOI) and search the LabCollector plasmids entries for tagging cassette plasmids using that marker. Some of these markers are flanked with direct repeats (DR) or loxP sites for recombination and excision of the marker at a subsequent step. The entry for each plasmid indicates which universal primers can be used to add a C-terminal tag to a gene with that plasmid.
  3. Gene Sequence:
    If there is not already a SnapGene file for your GOI in the DropBox directory “Sequence Files/Gene Sequences”, go to Saccharomyces Genome Database (http://yeastgenome.org). In the search window, enter the name of the gene of interest. Under the Sequence heading click the dropdown menu and select Genomic DNA +/- 1 kb. This will download a .fsa file, which you should open in SnapGene. Save the file in the Dropbox directory “Sequence Files/Gene Sequences” as a .dna (SnapGene) file, and rename it in the format GOI+/-1kb.dna. The GOI ORF is always oriented 5′-3′ in these files. Now find the ORFs and save the GOI ORF (starts at bp 1000) as a feature (don’t make it a common feature). Now save or export the file as a Standard GenBank (.gb) file in the same folder. Import the .gb file into LabCollector Sequences and give the entry the same name as the SnapGene file name. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to this entry. Link the Documents entry to the Sequence entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  4. Forward Protein C-terminal Tag Primer:
    Using the GOI sequence in SnapGene, select 60 bp within the ORF up to, but not including the STOP codon. Copy the sequence and paste it into a new SnapGene file. Now in the Dropbox directory “Sequence Files/KO or Tagging Universal Primers” open the SnapGene universal primer file suitable for performing a C-terminal tag/gene fusion cassette PCR amplification using the cassette plasmid you want to use. For example, the pFA6a cassette series uses the F2 primer and the pYM series uses the F3 primer. The suitable primers for a given cassette plasmid are listed in the LabCollector entry for each plasmid. Copy the sequence of the universal forward primer and paste it 3′ to the 60 bp gene-specific sequence. Now save the sequence as a SnapGene file (.dna) in the “Sequence Files/KO or Tagging Gene-Specific Primers” folder using the filename format GOI.universal primer name.dna format (e.g. CLN2.F2.dna). Use find common features (updated with primer sequences in the “Sequence Files/Export Standard Features” folder) to highlight the 3′ end of the primer sequence corresponding to the universal primer segment and save the file. Now save or export the file as a Standard GenBank file in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  5. Forward mRNA 3’UTR Tag Primer:
    Using the GOI sequence in SnapGene, select 60 bp within the ORF up to, and including the STOP codon. Copy the sequence and paste it into a new SnapGene file. Now in the Dropbox directory “Sequence Files/KO or Tagging Universal Primers” open the SnapGene universal primer file suitable for performing a 3’UTR mRNA cassette PCR amplification using the cassette plasmid you want to use. For example, the pDZ MS2SL and PP7SL cassettes use the m-TAGnew.for primer. The suitable primers for a given cassette plasmid are listed in the LabCollector entry for each plasmid. Copy the sequence of the universal forward primer and paste it 3′ to the 60 bp gene-specific sequence. Now save the sequence as a SnapGene file (.dna) in the “Sequence Files/KO or Tagging Gene-Specific Primers” folder using the filename format GOI_universal primer name.dna format (e.g. CLN2_m-TAG.for.dna). Use find common features (updated with primer sequences in the “Sequence Files/Export Standard Features” folder) to highlight the 3′ end of the primer sequence corresponding to the universal primer segment and save the file. Now save or export the file as a Standard GenBank file in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  6. Reverse Protein C-terminal Tag Primer:
    Using the GOI sequence in SnapGene, select 60 bp downstream of the ORF starting immediately after, but not including the STOP codon. Copy the sequence and paste it into a new SnapGene file, click the reverse complement box and save the file in the Dropbox directory “Sequence Files/KO or Tagging Gene-Specific Primers”. Open the SnapGene universal primer file suitable for performing a gene fusion cassette PCR amplification using the cassette plasmid you want to use. For example, the pFA6a and pYM cassette series use the R1 primer for C-terminal tag/gene fusions. The suitable primers for a given cassette plasmid are listed in the LabCollector entry for each plasmid. Copy the sequence of the universal reverse primer and paste it 3′ to the 60 bp gene-specific sequence (reverse complement of the copied sequence from the GOI sequence). Now save the sequence as a SnapGene file (.dna) using the filename format GOI.universal primer name.dna format (e.g. CLN2.R1.dna). Use find commoon features (updated with primer sequences in the “Sequence Files/Export Standard Features” folder) to highlight the 3′ end of the primer sequence corresponding to the universal primer segment and save the file. Now save or export the file as a Standard GenBank file in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  7. Reverse mRNA 3’UTR Tag Primer:
    Using the GOI sequence in SnapGene, select 60 bp downstream of the ORF starting immediately after, but not including the STOP codon. Copy the sequence and paste it into a new SnapGene file, click the reverse complement box and save the file in the Dropbox directory “Sequence Files/KO or Tagging Gene-Specific Primers”. Open the SnapGene universal primer file suitable for performing a 3’UTR mRNA fusion cassette PCR amplification using the cassette plasmid you want to use. For example, the pDZ cassettes use the m-TAGnew.rev primer for mRNA tags. The suitable primers for a given cassette plasmid are listed in the LabCollector entry for each plasmid. Copy the sequence of the universal reverse primer and paste it 3′ to the 60 bp gene-specific sequence (reverse complement of the copied sequence from the GOI sequence). Now save the sequence as a SnapGene file (.dna) using the filename format GOI_universal primer name.dna format (e.g. CLN2_m-TAG.rev.dna). Use find commoon features (updated with primer sequences in the “Sequence Files/Export Standard Features” folder) to highlight the 3′ end of the primer sequence corresponding to the universal primer segment and save the file. Now save or export the file as a Standard GenBank file in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  8. Forward Test Primers:
    Using the GOI sequence in SnapGene, select a sequence that is at least 65 bp upstream of the STOP codon (beyond the region of homology of the Forward tagging Primer) but no further than about 500 bp from the STOP codon. The sequence should have roughly 50-60% GC content, a melting temperature of 60-62° C, and the length should be between 20-24 bp. Make a note of the first nucleotide position relative to the START codon. Copy the sequence and paste it into a new SnapGene file. Save the file in the “Sequence Files/KO or Tagging Gene-Specific Primers” folder with the filename format GOI(number of nucleotides downstream of START codon)test.for (e.g. CLN2(400)test.for). Save or export the sequence as a Standard GenBank file (.gb) in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  9. Reverse Test Primers:
    Using the GOI sequence in SnapGene, select a sequence that is at least 65 bp downstream of the START codon (beyond the region of homology of the Reverse Tag Primer) but no further than about 500 bp from the STOP codon. The sequence should have roughly 50-60% GC content, a melting temperature of 60-62° C, and the length should be between 20-24 bp. Make a note of the first nucleotide position relative to the START codon. Copy the sequence and paste it into a new SnapGene file. Save the file in the “Sequence Files/KO or Tagging Gene-Specific Primers” folder with the filename format GOI(number of nucleotides downstream of START codon)test.rev (e.g. CLN2(1700)test.rev). Save or export the sequence as a Standard GenBank file (.gb) in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.
  10. Reverse Universal Test Primers:
    Most of the C-terminal tags and selectable markers will already have forward and reverse primers designed. For most cassettes, the marker gene is in the same orientation as the GOI and tag, so the reverse primer will be needed. If the C-terminal tag or marker do not already have primers, using the tag or marker sequence (from the plasmid sequence) in SnapGene, select a sequence within the C-terminal tag sequence or the selectable marker that is about 1 kb from the Forward Test Primer. The sequence should have roughly 50-60% GC content, a melting temperature of 60-62° C, and the length should be between 20-24 bp. Copy the sequence and paste it into a new SnapGene file, checking the reverse complement box. Save the file in the “Sequence Files/KO or Tagging Gene-Specific Primers” folder with the filename format TAG.rev or MARKER.rev (e.g. KanMX.rev). Save or export the sequence as a Standard GenBank file (.gb) in the “Sequence Files/Primers” folder. Import the primer sequence into LabCollector Sequences and name the entry the same as the primer name. Add a new Primer module entry with the same primer name and link it to the Sequence entry. Create a new Documents module entry with the same file name and upload the SnapGene (.dna) file as a link to the document. Link the Documents entry to the Sequence entry and the Primer entry. Link to the LabCollector Sequence entry when referring to it in ELN entries.The Forward Test Primer and Reverse Test Primer pair will produce a shart PCR product (~1 kb) if the GOI is not tagged (e.g. in wt control gDNA) and a much larger product (>2 kb) if the GOI is tagged. The Forward Test Primer is paired with a universal tag or marker primer (e.g. kanMX.rev) and produces a PCR product only if the GOI is tagged.

 

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