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When linearising a vector by restriction digest within the middle of a homologous region can a single cross over integration event only occur if the plasmid is re-ligated within the cell after transformation? If so, why do some organisms require transformation of a linearised vector rather than plasmid DNA?

An example: You amplify and clone GeneA into a vector containing an expression cassette. Before transformation, you digest this vector with a restriction enzyme at a site within GeneA to form a linear DNA fragment such as: GeneAStop_ExpressionCassette_StartGeneA.

My understanding is a double cross over event would occur in StartGeneA_ExpressionCassette_GeneAStop and the expression cassette would integrate between the two parts of the gene in the genome.

However, as the gene is cloned directly into the plasmid as one part and then digested to form a linear fragment, the expression_cassette is not in the region between two homologous fragments.

Does this mean a double cross over event cannot occur and a single cross over event would have to occur for the transformed DNA to integrate?

Can this single cross over event happen without relegation of the digested plasmid into circular DNA?

What would happen if you amplified by PCR rather than digested, as it would not be able to religate?

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  • $\begingroup$ Your post contains many different questions, so I responded to what I considered being the central question. If you were looking for a different answer, please focus your post such that the main question becomes clearer. $\endgroup$
    – gaspanic
    Commented Jun 3, 2022 at 22:54

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Single crossover is not the result of religation in the cell. The double-stranded break (DSB) in the plasmid instead stimulates a homologous recombination event, causing the entire plasmid to integrate into the homologous region in the chromosome. The result is two tandem copies of the homologous region, interspaced by the rest of the plasmid.

The figure below shows (1) a plasmid carrying a wild-type copy of the gene YFG1 (purple) and some selection marker AUXA (blue); and (2) a genome region with the gene yfg1 (pink) that is homologous to YFG1, but carries a mutation (black star). Arrows are shown for orientation.

After introducing a DSB in YFG1, this region will line up with homologous yfg1 region in the genome and integrate as shown. Note how the two tandem copies of YFG1/yfg1 resulting from the integration will both be "chimeras"; i.e., one part originates from the plasmid and the other from the chromosome. Also note how in this specific example, the mutation ends up in the second copy, since the DSB was introduced "upstream" of the mutation.

Single crossover example

Figure adapted from (1).

References and further reading

  1. Guthrie & Fink (1991): Guide to Yeast Genetics and Molecular Biology, Part A. pp 285-287. ISBN: 978-0-12-182095-4
  2. Orr-Weaver et al. (1981): Yeast transformation: A model system for the study of recombination. PMC349037
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