Rules for Designing FRT Primers

Eric Kofoid

Making in-frame deletions using FRT sites is straight forward. However, designing the primers can often be confusing. Here are some rules which will ease the process.

1. Some definitions



2. Upstream (left) primer:

Modular structure: [left homology block]—ATG—[1+3N]—FRT—[drug left]

The "left homology block" is identical to sequence preceeding the start site of gene X, the knock-out target. It can be extended to include the ATG and [1+3N] blocks and may be exactly these.

"ATG" is the start codon.

"[1+3N]" is a block of containing one plus a multiple of 3 nucleotides, and must be chosen not to introduce a nonsense triplet into the reading frame defined by the start codon (i.e., the 0 reading frame).

"FRT" is the corresponding sequence given above.

"drug left" is identical to a tract at the left end of a drug cassette and will allow PCR to initiate. It is generally 20 bases long.

3. Downstream (right) primer:

Modular structure: [right homology block]'—[stop]'—[3M]—FRT'—[drug PCR right]

"[right homology block]'" is identical to the complement of sequence following the stop codon of gene X. It can be extended to include the stop codon complement and the [3M] block and may be exactly these.

"[stop]'" is the complement of the stop codon.

"[3M]" is a block containing a multiple of 3 nucleotides. As before, these must be chosen not to introduce any complemented nonsense triplets into the 0 reading frame, defined in this case by the preceeding complemented stop codon. N and M need not be the same.

"FRT'" is the complemented FRT sequence given above.

"drug right" is identical to the complement of a tract at the right end of a drug cassette and will allow PCR to initiate. It is generally 20 bases long.

4. Product:

Modular structure: …ATG—[1+3N]—FRT—[3M]—[stop]…

The length is thus 3 + 1 + 3N + 35 + 3M + 3 = 3(14 + M + N).

This places the stop codon in frame with the start codon, and introduces no terminators from the FRT region. If care was taken with the design of the [1+3N] and [3M] blocks, the result is a minigene of 13 + M + N codons. Again, if the filler blocks were properly made, the peptide expressed will be identical to the wild-type protein only in the first 1+N and last M-1 amino acids.

5. Examples.

These primers were used to make an in-frame deletion of eutE:



1-3 = eutE start codon
1-40 = eutE homology block
41-75 = FRT
76-95 = camRL-core sequence.
In this case, N = 12.



1-3 = complemented eutE stop codon (TAA)
1-42 = complemented eutE homology block
43-77 = FRT'
77-96 = camRR-core sequence (note overlap with FRT').
In this case, M = 13.

Minigene (after FLP excision)


where underlines indicate the start and stop codons.

Peptide product


where underlines indicate regions common to EutE. In both cases, these are 1 amino acid longer than minimally expected, as the FRT seqeuence contributed leucine at each end. This residue is found, by chance, in the corresponding positions in EutE.

Last Update: Thursday June 19 2014
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