Short notes :
Shuttle vector : A shuttle vector is a vector (usually a plasmid) constructed so that it can propagate in two different host species. Therefore, DNA inserted into a shuttle vector can be tested or manipulated in two different cell types. The main advantage of these vectors is they can be manipulated in E. coli and then used in a system which is more difficult or slower to use (e.g., yeast, other bacteria).Shuttle vectors include plasmids that can propagate in eukaryotes and prokaryotes (e.g., both Saccharomyces cerevisiae and Escherichia coli) or in different species of bacteria (e.g., both E. coli and Rhodococcus erythropolis). There are also adenovirus shuttle vectors, which can propagate in E. coli and mammals. Shuttle vectors are frequently used to quickly make multiple copies of the gene in E. coli (amplification). They can also used for in vitro experiments and modifications (e.g., mutagenesis, PCR).
YAC : A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearised by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends. They were first described in 1983 by Murray and Szostak.
PCR : Polymerase chain reaction (PCR) is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations.
Electroporation : Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell’s function, or a piece of coding DNA.
Electroporation is a dynamic phenomenon that depends on the local transmembrane voltage at each cell membrane point. It is generally accepted that for a given pulse duration and shape, a specific transmembrane voltage threshold exists for the manifestation of the electroporation phenomenon (from 0.5 V to 1 v). This leads to the definition of an electric field magnitude threshold for electroporation (Eth). That is, only the cells within areas where E Eth are electroporated. If a second threshold (Eth) is reached or surpassed, electroporation will compromise the viability of the cells,
i.e.. irreversible electroporation.
