MOLECULAR BIOLOGY TECHNIQUES
The central foci of molecular biology are the nucleic acids, deoxyribonucleic acid (DNA) and the ribonucleic acid (RNA). Nucleic acids encode the genetic information specifying the primary structure of all proteins unique to an organism. Together with lipids and extracellular supporting stroma, they create cel-lular activity and physiological function. Thus, biological functions can be understood in part by examining the interrelationships between these key com-ponents. RNA and DNA are composed of four separate building blocks called nucleotides. Each of the four nucleotides contains a nucleic acid base (A, ad-enine; G, guanine; T, thymine; C, cytosine), a deoxyribose sugar moiety and a phosphoester for DNA. For RNA, the same bases as in DNA are present, except that uridine (U) is substituted for T and a ribose moiety is present instead of the deoxyribose. The nucleotides are connected one to another to form a chain-like arrangement, which comprises the nucleic acid’s sequence. RNA is composed of a single strand whereas DNA is composed of two paired strands. In order for the paired strands to match up, they must face each other in the opposite or complementary direction. The complementary strands of DNA are kept together primarily by the hydrogen bonds that form between the bases A and T (2 bonds) as well as C and G (3 bonds). It is this hydrogen bonding between the matched base pairs A and T (or, for RNA, A and U) as well as C and G that is the foundation of all molecular biological tests. Although one given base pair match of AT or GC would separate easily, there is strength in numbers and the more base pair matching, the greater the number of hydrogen bonds between the two DNA strands and the less likely they are to separate.
In the hybridization of nucleic acid strands, when two DNA strands meet, they orient each other in opposite or antiparallel directions to allow base pair matching to occur. If no base pair matching is present, they go their separate ways. However, if there is sufficient base pair matching they will join together of hybridize. The specific term used to describe the degree of base pair match-ing that determines if the strands stay together is homology. How much ho-mology is needed for two strands to stay together? Although it is true “the more, the better,” another important variable is how close the base pair matches are to one another. Adjacent base pair matches in a sequence will hold together more strongly than the same number of base pair matches dis-persed over the DNA sequence. Dispersed base pair matches are typical unre-lated DNA strands whereas clustered base pair matches are expected for re-lated complementary DNA molecules. Clearly, if two DNA strands are com-pletely homologous and have 100% base pair matching then the strands would tend to remain hybridized under most conditions. Conversely, hybridized strands with poor homology (e.g., only 10% of base pairs matched) would tend to dissociate or denature readily under most conditions. Whether hybridized strands with intermediate homology - where, for example, 50% of the base pairs matched - would remain hybridized would depend greatly on the reaction conditions.
Given that hydrogen bonds are the glue that keep two hybridized strands to-gether and that many chemicals and conditions can affect hydrogen bonding, a term is needed that describes whether the hybridization reaction conditions relatively favor or disfavor hydrogen bonding - stringency. Under low strin-gency conditions, hybridized strands with intermediate homology would tend to remain hybridized whereas hybridized strands with poor homology would dissociate. At high stringency conditions, only hybridized strands with strong homology would tend to remain hybridized.
Another key term, the melting temperature, or commonly abbreviated, Tm. If one takes two strands of DNA that share homology and hybridizes them, at any given time some of the strands will remain hybridized whereas others will have separated. The ratio of hybridized/denatured DNA strands in the reaction will vary depending on the degree of homology as well as any condition that may affect hydrogen bonding between matched base pairs such as formamide con-centration and temperature. The melting temperature is defined as that tem-perature under the specific reaction conditions where one half of the hybrid-ized strands are still hybridized and the other half are denatured.
Two molecular biology-based techniques discussed here are: gene probe and polymerase chain reaction (PCR).
Copyright © 2018-2020 BrainKart.com; All Rights Reserved. Developed by Therithal info, Chennai.