Process for the rescue of DNA and for detecting mutations in mar

Details Process for the rescue of DNA and for detecting mutations in mar Process for the rescue of DNA and for detecting mutations in mar

1993-01-29

1995-11-28

Jones, W. Gary

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 4351721, 4351723, 435243, 435244, 536 221, 536 231, 935 19, 935 23, 935 29, 935 56, 935 57, 935 83, 935 84, C12Q 168

Patent

active

054707064

ABSTRACT:
The invention relates to a process for the rescue and cloning of a DNA sequence wherein a vector containing said DNA sequence is multiplied in a suitable bacterial host. As the bacterial host, a bacterial strain which is incapable of host restriction is used, such as an Escherichia coli C strain. Preferably, a bacteriophage vector which is a suitable cloning vehicle for the bacterial host concerned is used as the vector, the step of in vitro packaging into phage coats being performed by means of a phage packaging extract obtained from a bacterial strain which is incapable of host restriction. The invention may be used in a process for detecting mutations in one or more marker genes introduced into a mammalian genome by means of a vector, which process comprises isolating DNA from cells of the transgenic mammal or from the mammalian cells, recovering the vector from said DNA, multiplying the vector in a suitable bacterial host which is deficient with respect to at least one of the marker genes, and testing marker gene expression. Preferably, the DNA isolated from cells of the transgenic mammal or the mammalian cells is pre-purified by fragmenting the DNA by means of a restriction enzyme which does not have a cutting site within the vector, separating the resulting fragments on the basis of a suitable criterion, such as differences in size, and collecting fragments comprising the vector, whereafter the vector is recovered from said pre-purified DNA.

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