Genetic Manipulation - Environmental Biotechnology: Theory and Application - page 216

based on making the membrane permeable to the DNA molecule. However, if
the 'foreign' DNA is part of a recombinant virus, it has to be packaged into
particles, and then transferred into cells by infection. A check may be made on
the product by carrying out analyses described later.
DNA for transfer
Most commonly, this is a piece of double stranded DNA which contains the
coding sequence for a gene. It may have been obtained from a number of sources,
for example genomic DNA, a complementary DNA (cDNA) library, a product
of a polymerase chain reaction (PCR) or a piece of DNA chemically produced
on a DNA synthesiser machine. Another source is from a DNA copy of an RNA
virus as in the replicative form of RNA viruses.
Genomic libraries
Genomic DNA, in this context, is material which has been isolated directly from
an organism, purified and cut up into pieces of a size suitable to be inserted into
a cloning vector. These pieces may either be ligated as a total mixture, into a
suitable vector to produce a genomic library, or a specific piece may be isolated
and prepared as described above. Genomic libraries are very useful, as they
may be amplified, and accessed almost limitlessly, to look for a specific DNA
sequence thus reducing the amount of work involved in any one experiment. The
disadvantage is that if the genomic library is of a eukaryotic origin, which is
almost exclusively the case, the genes will contain regions, or introns, which are
quite normally inserted along its length and are processed out of the RNA copy
during maturation prior to protein synthesis. This is a problem if the gene is to be
expressed, in other words, if the protein is to be made from the DNA blueprint.
Prokaryotics do not contain introns in their genes and so do not possess the
mechanisms for their removal. Furthermore, introns are not necessarily processed
correctly even if the expression system is eukaryotic. This problem can be avoided
by using a cDNA instead of a genomic library.
cDNA libraries
In eukaryotes, the first product of transcription from DNA is not messenger RNA
(mRNA) but heterogeneous nuclear RNA (hnRNA). This is mRNA prior to the
removal of all the non-coding sections, or introns, which are discarded during
the processing to produce the mature mRNA. cDNA is DNA which has been
artificially made using the mature mRNA as a template, which is then used as
the template for the second strand. Thus the synthetic DNA product is simply a
DNA version of the mRNA and so should overcome the problems of expression
outlined above.