In an attempt to clone a gene into the TOL plasmid, it was observed that the restriction product of the gene of interest were blunt ended, thereby reducing the ligation efficiency of this gene and the TOL plasmid. Elucidate two methods that could be employed to increase the ligation efficiency of your restriction products.
Bacterial Genomics
The study of the morphological, physiological, and evolutionary aspects of the bacterial genome is referred to as bacterial genomics. This subdisciplinary field aids in understanding how genes are assembled into genomes. Further, bacterial or microbial genomics has helped researchers in understanding the pathogenicity of bacteria and other microbes.
Transformation Experiment in Bacteria
In the discovery of genetic material, the experiment conducted by Frederick Griffith on Streptococcus pneumonia proved to be a stepping stone.
Plasmids and Vectors
The DNA molecule that exists in a circular shape and is smaller in size which is capable of its replication is called Plasmids. In other words, it is called extra-chromosomal plasmid DNA. Vectors are the molecule which is capable of carrying genetic material which can be transferred into another cell and further carry out replication and expression. Plasmids can act as vectors.
In an attempt to clone a gene into the TOL plasmid, it was observed that the restriction product of the gene of interest were blunt ended, thereby reducing the ligation efficiency of this gene and the TOL plasmid. Elucidate two methods that could be employed to increase the ligation efficiency of your restriction products.
Restriction Enzymes or “Restriction endonucleases” are proteins that cut DNA at a specific base sequence. Restriction enzymes produce either a sticky-end or blunt-end DNA fragment.
EcoRI is a restriction Enzyme that can be used to produce sticky ends with a 5′ overhang. It recognizes the six-base sequence 5′ GAATTC 3′,
HaeIII, and EcoRV restriction Enzyme can be used to produce blunt ends. HaeIII recognizes and cuts the DNA at the positions where the GGCC sequence is found.
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