Understanding DNA Linkers, Adapters, and Selection of Recombinants
DNA linkers and adapters play crucial roles in genetic engineering by facilitating ligation of DNA molecules and selecting appropriate restriction enzymes. Furthermore, DNA hybridization allows for the assessment of evolutionary relatedness based on base pair sequences. This process involves the formation of hybrid double helices between DNA strands from different species.
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Linkers and selection of recombinents Dr,M.Deivanayaki
An adapter or adaptor, or a linker in genetic engineering is a short, chemically synthesized, single-stranded or double-stranded oligonucleotide that can be ligated to the ends of other DNA or RNA molecules. Double stranded adapters can be synthesized to have blunt ends to both terminals or to have sticky end at one end and blunt end at the other. For instance, a double stranded DNA adapter can be used to link the ends of two other DNA molecules (i.e., ends that do not have "sticky ends", that is complementary protruding single strands by themselves). It may be used to add sticky ends to cDNA allowing it to be ligated into the plasmid much more efficiently. Two adapters could base pair to each other to form dimers. A conversion adapter is used to join a DNA insert cut with one restriction enzyme, say EcoRl, with a vector opened with another enzyme, Bam Hl. This adapter can be used to convert the cohesive end produced by Bam Hl to one produced by Eco Rl or vice versa.
SELECTION OF RESTRICTION ENZYMES When selecting restriction enzymes, you want to choose enzymes that: Flank your insert, but do not cut within your insert. Are in the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmid.
DNA HYBRIZATION Evolution deals with heritable changes in populations over time. Because DNA is the molecule of heredity, evolutionary changes will be reflected in changes in the base pairs in DNA. Two species that have evolved from a common ancestor will have DNA that has very similar base pair sequences. The degree of relatedness of two species can be estimated by examining how similar their base pair sequences are. One method of assessing relatedness uses hybridization of DNA. In the molecular genetic technique of hybridization of DNA, single strands of DNA from two different species are allowed to join together to form hybrid double helices. These hybrid segments of DNA can be used to determine the evolutionary relatedness of organisms by examining how similar or dissimilar the DNA base pair sequences are. The technique of DNA hybridization is based on two principles: the first, that double strands of DNA are held together by hydrogen bonds between complementary base pairs, and the second is that the more closely related two species are, the greater will be the number of complementary base pairs in the hybrid DNA. In other words, the degree of hybridization is proportional to the degree of similarity between the molecules of DNA from the two species. Hybridization of DNA is accomplished by heating strands of DNA from two different species to 86 C [186.8 F]. This breaks the hydrogen bonds between all complementary base pairs. The result is many single-stranded segments of DNA. The single-stranded DNA from both species is mixed together and allowed to slowly cool. Similar strands of DNA from both species will begin to chemically join together or re-anneal at complementary base pairs by reforming hydrogen bonds.
The resulting hybrid DNA is then reheated and the temperature at which the DNA once again becomes single-stranded is noted. Because one cannot observe DNA separating, another technique must be used simultaneously with heating to show when separation has occurred. This technique employs the absorption of UV light by DNA. Single strands of DNA absorb UV light more effectively than do double strands. Therefore, the separation of the DNA strands is measured by UV light absorption; as more single strands are liberated, more UV light is absorbed. The temperature at which hybrid DNA separation occurs is related to the number of hydrogen bonds formed between complementary base pairs. Therefore, if the two species are closely related, most base pairs will be complementary and the temperature of separation will be very close to 86 C [186.8 F]. If the two species are not closely related, they will not share many common DNA sequences and fewer complementary base pairs will form. The temperature of separation will be less than 86 C [186.8 F] because less energy is required to break fewer hydrogen bonds. Using this type of information, a tree of evolutionary relationships based on the separation temperature of the hybrid helices can be generated.
