Quiz 3: Recombinant Dna Technology and Genomics
The terms recombinant DNA (deoxyribonucleic acid) technology, gene cloning, and genetic engineering are used to describe the same technique. These techniques differ slightly in their methodologies that are interrelated. Gene cloning can be defined as the replication of fragments of deoxyribonucleic acid (DNA) with the help of a genetic material, which is self-replicating. Gene cloning duplicates only single genes of the DNA of an organism, unlike the reproductive cloning, in which the entire organism is replicated. The recombinant DNA technology technically involves the combining of fragments of DNA from different sources, whereas the manipulation of alteration of the genetic composition of an organism is involved in genetic engineering. The example of the recombinant DNA technology is the ligation of a piece of human DNA in to the bacterial chromosome. The example of genetic engineering is to place the piece of recombinant DNA formed by using the recombinant DNA technology into a bacterial cell to create a bacterium.
The importance of DNA ligase enzyme in the RDT or recombinant DNA experiments is that it is used to form a phosphodiester bonds between the fragments of the DNA. The formation of phosphodiester bond is an important step in the experiments of cloning because between the sticky ends of the fragments of the DNA the hydrogen bonds are not strong enough to hold a recombinant DNA molecule together for long time or permanently. The restriction enzymes are the enzymes that cut the DNA at recognition sequences or specific nucleotide sequences. The restriction enzymes produce two types of ends in DNA fragments, sticky ends and blunt ends. Often, the restriction enzymes are used to digest the DNA into fragments as an important step in many experiments of cloning. The restriction enzymes are used before the use of DNA ligase enzyme to join the fragments of DNA together.
The cloned sequence of a gene that thought to be responsible for controlling the fertilizing capability of the rat sperm could be used to synthesize primers that probably can be used to amplify the deoxyribonucleic acid (DNA) from the human cells in an attempt to amplify the complementary genes found in humans. If the desired polymerase chain reaction (PCR) products were successfully obtained from the above experiment, the sequencing of PCR products could be done and also compared to the rat gene to search for sequences of nucleotide which are similar. This would suggest that these genes are related or not. Also, in library screening experiments these polymerase chain reaction (PCR) products could be used as probes to find or search the full-length human gene. In northern blot analysis, these PCR products can also be used as probes to determine if the mRNA (messenger ribonucleic acid) for this gene is expressed in tissues of humans also.
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