Recombinant DNA technology has been a mainstay to modern drug discovery for decades now and will continue to be a vital component of the field aforementioned. The basis of recombinant DNA technology is the ability to manipulate DNA molecules in a test tube. It utilizes purified enzymes whose activities are known and can be controlled to make specified changes to the DNA molecules that are being manipulated.
Basically, what science majors need to know about recombinant DNA covers a wide range of aspects. Firstly, it is essential for science majors to know the enzymes that are used in recombinant DNA, as they are the central for how recombinant DNA works. These enzymes fall into four broad categories and will be described and elaborated below.
(i) DNA polymerases
Many of the techniques used to study DNA depend on the synthesis of copies of all or part of existing DNA or RNA molecules. DNA polymerase makes new DNA polynucleotides whose sequence is dictated, via the base pairing rules, by the sequence of nucleotides in the DNA molecule that is being copied. In order to initiate DNA synthesis there must be a short double-stranded region to provide a 3′ end onto which the enzyme will add new nucleotides.
DNA polymerases can have two types of activity, namely the 3′-5′ exonuclease activity or the 5′-3′ exonuclease activity. The 3′-5′ exonuclease activity enables the enzyme to remove nucleotides from the 3′ end of the strand that it has just synthesized. This is called the proofreading activity because it allows the polymerase to correct errors by removing a nucleotide that has been inserted incorrectly and subsequently insert the correct nucleotide. In contrast, the 5′-3′ exonuclease activity is possessed by some DNA polymerases whose natural function in genome replication requires that they must be able to remove at least part of a polynucleotide that is already attached to the template strand that the polymerase is copying.
(ii) Nucleases
The main function of nucleases is to degrade DNA or RNA molecules by breaking the phosphodiester bonds that link one nucleotide to the next. This particular enzyme enables prokaryotic and eukaryotic DNA to be combined, which is an important property in producing proteins such as insulin. The nucleases have two broad classifications: the exonucleases and the endonucleases. The exonucleases cleave nucleotides from the end of DNA molecules. On the other hand, endonucleases cleave internal phosphodiester bonds. They do this by recognizing short DNA sequences that are generally 4 to 8 base pairs in length that are often inverted repeats. The endonucleases may create blunt or sticky ends on DNA molecules.
(iii) Ligases
Ligases join DNA molecules together by synthesizing phosphodiester bonds between nucleotides at the ends of two different molecules. In recombinant DNA technology, DNA ligase is used to covalently join restriction fragments in vitro. The Bacteriophage T4 enzyme is most commonly used. In short, DNA ligases ligates blunt or sticky ends of DNA molecules.
(iv) End-modification enzymes
These enzymes function to make changes to the ends of DNA molecules.
Secondly, science majors need to know about the polymerase chain reaction (PCR) to have a solid understanding of recombinant DNA, as it is PCR that revolutionized molecular biology. PCR is a rapid and versatile in vitro method for amplifying defined target DNA sequences present within a source of DNA. They are designed to permit selective amplification of specific target DNA sequence(s) within a heterogeneous collection of DNA sequences. The selectivity is dependent upon how well the PCR is designed. Some prior DNA sequence information from the target sequences is required.
The requirements for PCR include:
– DNA template comprising of the genomic DNA and the cDNA (copy of mRNA)
– Synthetic DNA primers (20 to 30 bases) with one in the forward direction and one reverse; determines the selectivity of the amplification
– Nucleotides (ATP, GTP, TTP, CTP)
– Buffer containing magnesium
– Thermophilic DNA Polymerase
Thirdly, science majors need to know about expression vectors as they are an important part of recombinant DNA as well. Basically, expression vectors are used to produce a large amount of protein. Expression vectors contain a highly active promoter that results in large amounts of mRNA being produced. There is a wide range of vectors for bacteria, yeast, insects and mammalian cells; selection of which vector to use should be guided by multiple factors.
Plasmids are among the most commonly used vectors. They are double-stranded, closed circular DNA molecules ranging in size from 1 kilobasepair to 200 kilobasepairs. Plasmids contain a selectable marker to allow for rapid isolation of cells containing the plasmid, and the most common is the antibiotic resistance gene(s) for antibiotics such as ampicillin, tetracycline and puromycin. Plasmids are maintained extrachromosomally in bacteria, and the plasmids from Escherichia coli is most commonly used. The DNA sequence can be easily modified in a plasmid.
Finally, it would certainly be an advantage for science majors to know about some of the products of recombinant DNA, such as recombinant insulin and recombinant erythropoietin (EPO). Recombinant insulin in produced from Escherichia coli and its structure is absolutely identical to that of the endogenous molecule. Recombinant EPO, on the other hand, is produced in Chinese hamster ovary cells. However, glycosylation is not identical to endogenous EPO.
Reference:
1. Recombinant DNA Technology Lecture, Molecular and Chemical Basis of Therapeutics 300.
