Identification of transformants. In most cloning experiments the major part of the programme will be concerned with the identification of the transformed recombinant clones. Normally the quantity of transforming DNA incorporated into the recipient host will be very much less than the host chromosomal DNA, so identification of product expressed from the transforming fragment will be extremely difficult. However,various methods have been developed to identify clones that carry desired DNA sequences.
(a)Complementation Some in-coming genes are able to complement defective copies of the same gene on the chromosome of the recipient: that is,they code for a normal, active product for which the recipient is deficient. For example, the LACZ gene(coding for galactosidase) will complement a lacz ,galactosidase-deficient host to yield lac recipients which can be selected as red colonies on MacConkey's Agar.
(b)Antibiotic resistance genes These genes confer resistance to an antibiotic in normally susceptible cells. This can be exemplified with plasmid pBR322 which carries genes for tetracycline and ampicillin resistance and transformants can be selected on media containing one of these antibiotics:the foreign DNA is usually cloned into the gene coding for resistance to the other,unselected antibiotic.
(c)Hybridization A widely used method of analysing a collection of recombinant DNA molecules or cloned cells is by colony or plaque hybridization. Cells containing recombinant DNA are transferred to a nitrocellulose filter and lysed. Purified,complementary radioactive nucleic acids are then hybridized to the DNA in the nitrocellulose-bound cells. Complementary DNA will hybridize and it can be detected by autoradiography. The extent of hybridization will reflect the amount of complementarity. Copy DNA synthesized from an mRNA template can also be used to screen for recombinant DNA bound to a nitrocellulose filter in a similar way.
(d)Restriction endonuclease analysis This method can be used to determine whether the cloned DNA produces the same size of fragments as a standard DNA molecule. Restriction endonulease analysis provides mapped fragments which are often a prerequisite to sequence analysis.
(e)Immunological methods Immunological methods are finding increasing use and depend largely on the development of antibodies, primarily against protein products.
Identification of recombinant DNA is a highly secretive aspect of industrial cloning programme and the results of most of the studies will remain privileged information. This is an inevitable but regrettable aspect of the commercial importance of these new technologies.
Genetic engineering must be properly seen as the capstone of the massive framework of applied genetics which has been developed so successfully over the past three decades. Notwithstanding, it is certainly the most exciting and potentially the most creative technique available to the industrial geneticist.
The scientific and economic applications of recombinant DNA technology are limitless and will include:
(1)Increasing the yield of a particular gene product.
(2)Improving the rate of synthesis of a particular end product by cloning additional enzymes into the organism or by specifically altering a cloned enzyme by site-directed mutation(e.g. cloning energy-conserving nitrogen pathway from E.coli into ICI single cell protein organism Methylophilus methylotrophus).
(3)"Tailoring" an organism by transferring a particular activity to a more desirable host organism.