Cloning and DNA Recombination In Biotechnology

Nama : Arya Wira Wardhana

Npm : 21025010035

Class : A

Cloning and DNA Recombination

Once it was known that DNA molecules are genetic material that determines the properties of an organism, and that bacterial cells can spontaneously accept foreign DNA, several researchers immediately conducted research to manipulate the genetic properties of several types of cells. Various studies have been conducted to insert foreign DNA into bacterial cells, fungal cells, plant cells and animal cells. However, in the first stage of the manipulation many experienced failure. This is because only a few species of bacteria can spontaneously accept DNA. In addition, foreign DNA that has successfully entered the cell can only survive if it can replicate autonomously, or can be integrated into the host chromosome. Generally, foreign DNA that enters the chromosomal DNA will be immediately degraded by nuclease enzymes contained in the host cell. (Radji, M., 2011) Genetic modification of a new organism can be done in line with the discovery and development of various techniques in molecular biology. These include DNA isolation and purification techniques, the discovery of restriction endonuclease enzymes, DNA polymerase and DNA ligase enzymes, the discovery of plasmid DNA and DNA transfer techniques, DNA detection techniques, gene mapping techniques, and cultivation techniques. (Radji, M., 2011)

DNA molecules that are often used in recombinant DNA technology are plasmid DNA and genomic DNA derived from bacterial cells. Basically, the isolation of total genomic DNA from bacterial cells consists of several stages, namely:1.Cell cultivation in appropriate media2.Breakdown of the cell wall 3.Genomic DNA extraction 4.Purification of DNA Bacterial cell wall breaking is done physically, for example by sonication, or chemically by using the enzymes lysozyme, ethylene diamin tetraacetate (EDTA), or a combination of both. Under certain conditions, cell wall breakdown is sufficient with lysozyme and EDTA, but other materials that can lyse the cell wall are often added, such as triton X-100 detergent or sodium dedosyl sulfate (SDS). After cell lysis, the next step is to separate cell debris by centrifugation. Insoluble cell components are precipitated by centrifugation so as to leave the cell extract in a clear supernatant. (Radji, M., 2011; Thermo Scientific, 2009) The final stage of DNA isolation is the DNA purification process. Besides DNA, cell extracts contain large amounts of protein and RNA. Generally, DNA purification is done by adding phenol solution or a mixture of phenol and chloroform in a ratio of 1: 1, to precipitate proteins by centrifuging and enzymatically destroyed with proteinase. DNA that has been cleaned from proteins is still mixed with RNA so it is necessary to add RNAse to clean DNA from RNA. DNA molecules that have been isolated are thenpurified by "ethanol precipitation". In the presence of a solution of garam solution (monovalent cations such as Na+), at a temperature of -20oC absolute ethanol can precipitate DNA well so that it is easily separated by centrifugation. (Radji, M., 2011; ThermoScientific, 2009)

Isolation and purification of plasmid DNA from bacterial cells is basically the same as the isolation of genomic DNA. Bacterial cells containing plasmid DNA are cultured and harvested. Bacterial cells are lysed with the addition of detergent and lysozyme enzyme, then centrifuged to separate cell debris from the cell extract. The next process is to separate proteins and RNA from plasmid DNA. However, there are important differences in plasmid DNA isolation with genomic DNA isolation. Plasmid DNA isolation pays attention to the presence of genomic DNA derived from bacterial cells. The separation between plasmid DNA and genomic DNA is very important if plasmid DNA is to be used as a vectorkloning. Even a small amount of bacterial genomic DNA contamination can affect the success of DNA cloning. (Radji, M., 2011; Thermo Scientific, 2009) Several ways to remove genomic DNA in plasmid DNA purification have been developed. How to separate plasmid DNA with genomic DNA is principally based on its size and conformation. The size of plasmid DNA is very small when compared to the size of genomic DNA. The size of the largest plasmid DNA is less than 8% of the size of bacterial genomic DNA, and most plasmid DNA is smaller than that size. Thus, techniques that can separate small DNA molecules with large DNA will be very effective for separating plasmid DNA. (Radji, M., 2011)

Different restriction endonuclease enzymes have different cutting sites, but there are several types of restriction endonuclease enzymes isolated from different sources that have the same cutting site. Restriction endonuclease enzymes that have the same cutting site are called isochizomer . (Radji, M., 2011) The DNA base sequence at the cutting site has the same base sequence on the double helix DNA strand, known as a palindromic sequence. palindromic sequences  For example, the EcoRI enzyme, which was first isolated by Herbert Boyer in 1969 from Escherichia coli which cuts DNA in the section between bases G and A in the GAATTC sequence. The results of cutting restriction endonuclease enzymes there are two kinds that produce blunt ends blunt ) and sticky end (sticky) or cohesive. (Radji, M., 2011)

The image below shows, for example, the restriction enzyme EcoRI, cutting a DNA molecule at a sequence of hexa- nucleotide sequence 5'- GAATTC -3' at the position between bases G and A. Similarly, the polychromic sequence 3'- CTTAAG -5' the enzyme Eco RI enzyme, also cuts at a position between bases A and G. Thus the double helix DNA molecule that is cut by the EcoRI enzyme, produces a restriction fragment with sticky ends. Some types of enzymes, such as AluI, produce blunt restriction fragments because they cut double helix DNA right in the middle between the C and G bases. Currently, many restriction endonuclease enzymes have been purified and commercially produced that can recognize different nucleotide sequences.