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DNA for transfer
Most commonly, this is a piece of double-stranded DNA which contains the cod-ing sequence for a gene. It may have been obtained from a number of sources, for example, genomic DNA, a cDNA library, a product of a polymerase chain reaction (PCR) or a piece of DNA chemically produced on a DNA synthe-siser machine. Another source is from a DNA copy of an RNA virus as in the replicative form of RNA viruses.
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 in 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. Prokaryotes 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.
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 noncoding sections, or introns, which are discarded during the processing to produce the mature mRNA. Complementary DNA (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.
The polymerase chain reaction (PCR) is a powerful technique which amplifies a piece of DNA of which only a very few copies are available. The piece must be flanked by DNA whose sequence is known or at least a close approximation can be guessed. This knowledge allows a short sequence of DNA to be synthesised of only a few nucleotides long, to bind specifically to the end of the sequence and act as a primer for the DNA polymerase to make one copy of the whole piece of DNA. A second probe is used for the other end to allow the second strand to be synthesised. The process is repeated by a constant cycling of denaturation of double-stranded DNA at elevated temperature to approximately 95 ◦ C, followed by cooling to approximately 60 ◦C to allow annealing of the probe and comple-mentary strand synthesis. This technique requires the use of DNA polymerases able to withstand such treatment and two bacteria from which polymerases have been isolated for this purpose are Thermococcus litoralis and Thermus aquaticus.
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