Peccoud Lab Protocol: how to design primers for generating a de novo gene deletion in yeast

Introduction

Yeast strains carrying single deletions of non-essential genes are commercially available from GE Life Sciences (http://dharmacon.gelifesciences.com/cdnas-and-orfs/non-mammalian-cdnas-and-orfs/yeast/yeast-knockout-collection/). However, these are available only as deletions in which the gene is replace with the kanMX selectable marker (G418 resistance). It is sometimes necessary to generate deletion strains de novo by gene replacement with a PCR amplicon of a selectable marker flanked by 5′ and 3′ DNA sequences of 40-60 bp homologous to the flanking DNA on either side of the ORF to be replaced/deleted. This protocol describes how to design pairs of primers to make de novo yeast gene deletion strains.

Method

1. Check LabCollector for Existing Stocks of the Desired Primers Before Starting the Design Process

2. Plasmid Sequence:

Decide which selectable marker you wish to replace the gene of interest (GOI) with and search the LabCollector plasmids entries for knockout 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, allowing one to re-use the marker in additional gene deletions in the same strain. The entry for each plasmid indicates which universal primers can be used to perform gene deletions 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 KO Primer:

Using the GOI sequence in SnapGene, select 60 bp upstream of the ORF up to, but not including the START 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 gene deletion cassette PCR amplification using the cassette plasmid you want to use. For example, the pFA6a cassette series uses the F1/univF2 primer for gene deletions. 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.F1.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.

5. Reverse KO 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 deletion cassette PCR amplification using the cassette plasmid you want to use. For example, the pFA6a cassette series uses the R1 primer for gene deletions. 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.

6. Forward Test Primers:

Using the GOI sequence in SnapGene, select a sequence that is at least 65 bp upstream of the START codon (beyond the region of homology of the Forward KO Primer) but no further than about 1 kb from the START 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 upstream 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.

7. Reverse Test Primers:

Using the GOI sequence in SnapGene, select a sequence within the ORF of the GOI 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. Make a note of the last nucleotide position relative to the START codon. 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 GOI(number of nucleotides downstream of START codon)test.rev (e.g. CLN2(460)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.

8. Reverse Universal Test Primers:

Most of the 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 marker does not already have primers, using the marker sequence (from the plasmid sequence) in SnapGene, select a sequence within the selectable marker that is about 500 bp from the start or end of the marker cassette (including the promoter for reverse primers or the terminator for forward primers). 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 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 PCR product (~1 kb) only if the GOI is not deleted (e.g. in wt control gDNA). The Forward Test Primer is paired with a universal marker primer (e.g. kanMX.rev) and produces a PCR product only if the GOI is replaced with the selectable marker.