Isolation of Nucleic Acids

Isolation of genetic material is important in the study of recombinant DNA technology, forensics and medicine (University of Utah 2011). Procedures utilized in the isolation of DNA depend on the source, age and size of sample. Though different reagents may be utilized in the isolation of genetic material from the specific biological sources, all protocols consist of a general procedure: (1) cell lysis, (2) DNA purification and (3) DNA isolation.

Cell lysis can be achieved through physical or chemical disruption; both were utilized in the experiment through homogenization of the liver and utilization of sodium dodecyl sulphate (SDS) solution. Lysis is usually carried out in a salt solution to aggregate protein and cellular debris, saline-ethylenediaminetetraacetic acid (EDTA) was utilized in the experiment. EDTA is a chelating agent that binds to magnesium and calcium ions that are necessary in the action of DNase (World of Forensic Science 2006). The solution usually contains a detergent, in this case SDS, which disrupts the cell membrane through the denaturation of proteins (University of Utah 2011). Heat can promote lysis of cells though subsequent cooling is essential to ensure integrity of the DNA.

DNA purification can be achieved through the use of chemicals, particularly: phenol, chloroform and isoamyl alcohol. Phenol disassociates proteins from DNA; the nonpolar solution causes a change in the polarity of proteins. The less-polar residues of the amino acids (usually surrounded by polar residues in vivo) fold out making it soluble in the nonpolar phenol (Oswald 2008). Chloroform denatures proteins and lipids and it also makes DNA less soluble to phenol; it is usually mixed with isoamyl alcohol to prevent reaction with oxygen. Isoamyl alcohol also reduces foaming (Moors 2011).

DNA isolation can be achieved through alcohol precipitation. Ethanol or isopropyl alcohol can be utilized in the procedure. The nonpolar nature of the two solutions makes it possible for the aggregation of the polar DNA (Moors 2011). With the isolated genetic material further analysis can be utilized, including restriction enzyme digestion and agarose gel electrophoresis.

Restriction enzymes are isolated from particular strains of bacteria like Escherichia coli, Haemophilus influenza, Bacillus amyloliquifaciens among others. DNA sequences recognized by restriction enzymes are symmetrical along an axis (Pearson Education Inc. 2007), cleavage of DNA is not necessarily along the axis but depends on enzyme utilized (Refer to Fig. 3) Cleavage occurs through hydrolysis; cleaved DNA can correlate through condensation reactions (Rothman 2011).

HindIII is a type II restriction enzyme that recognizes and cleaves the palindromic sequence AAGCTT (Refer to Figure 4). Isolated from H. influenza, the restriction enzyme cleaves DNA near or at the recognized sequence in the presence of magnesium ions (Tang et. al 2011).

Agarose gel electrophoresis (AGE) utilizes electricity to separate DNA fragments according to size. As the current passes through the