Re: what does cloning in frame mean? and the importance of it.

Hi Ash,

Searching our archives can give you some background information on protein translation and codons and such, so I'll start by simply stating that genetic sequences are read into protein sequences by assigning one amino acid for every three nucleotide bases (one codon). Since the sequence is read in threes, where you start reading will determine where the codons will be placed:

GATGGATTACAAGGATGACGATGACAAGCATG...
 |  |  |  |  |  |  |  |  |  |
AspGlyLeuGlnGly***

  |  |  |  |  |  |  |  |  |  |
 MetAspTyrLysAspAspAspAspLysHis...

   |  |  |  |  |  |  |  |  |  |
  TrpIleThrArgMetThrMetThrSerMet...

During normal protein synthesis, the frame is determined by the start codon, usually the first ATG (Met) in the sequence, such that frame isn't an issue when cloning entire sequences. The problem of frame comes into play when you are cloning two genes together to make a fusion protein. In this case, the reading frame of the upstream gene has to match the reading frame of the downstream gene, since the start codon of the upstream gene will determine the frame for the entire sequence. For example, if the sequence above were placed downstream of gene in the first frame, the TGA stop codon would terminate the read before the rest of the gene was included. Similarly, if another gene were placed downstream of this sequence in the third frame, it would be misread because the ATG in the second frame comes before the ATG's in the third frame. To overcome this potential hazard, most commercially available fusion protein vectors are constructed and sold in all three frame.

Hope this helped,
Mike