⇒ Recombinant DNA Technology
⇒ Restriction Endonucleases
⇒ Host Cells
A number of bacterial and yeast strains have been developed for recombinant
DNA experiments. In order for a given plasmid to be replicated by
a host cell, the cell must recognize its origin of replication site (oriC). Recombinant
plasmid vectors are normally introduced into competent cells
by transformation and then selected using appropriate cell culture media.
For example, if the vector contains an ampR
gene that encodes resistance
to ampicillin, the culture media would include that antibiotic to ensure
that only transformed cells will grow.
Another method for introducing recombinant DNA molecules into
host cells is electroporation. In this method, a suspension of exponentially
growing host cells is mixed with a solution of recombinant DNA
molecules and exposed to a high electric field for a few milliseconds. The
high voltage alters the structure of the membrane so that pores are temporarily
formed, allowing plasmid DNA to enter the cell. This method is
fast and efficient.
If bacteria are used as the host to clone eukaryotic genes, certain
steps must be taken to make it possible for the bacteria to make sensible
mRNAand functional proteins, since bacteria do not possess mechanisms
for processing eukaryotic pre-mRNA molecules. To do this, it is necessary
to isolate already processed mRNA from the donor eukaryotic cells
and convert the single-stranded RNA to double-stranded DNA . Reverse
transcriptase from retroviruses uses RNAtemplates for
synthesizing DNA . The resulting DNA molecules, known as cDNA (for
complementary DNA ), can then be used for cloning in bacteria since they
posses only intron-free protein-coding genetic information.
Common hosts are E. coli and S. cerevisiae.