In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty expanded on the earlier work of Fredrick Griffith, who discovered the process of transformation in 1928. Avery, MacLeod, and McCarty investigated the chemical nature of this transforming factor - more specifically, whether the transforming factor was a protein or a nucleic acid. They attempted to transform the R strain into the S strain by incubating living R and heat-killed S. However, they pretreated the heat-killed S with either a protease (an enzyme that degrades proteins) or with DNAase, and enzyme that degrades DNA. They reasoned that if the transforming factor was a protein, treatment of the heat-killed S with a protease would destroy the protein and inhibit transformation and treatment with DNAase should have no effect on the transformation process. On the other hand, if DNA were the genetic material, the opposite would be true. In their experiments, Avery found that protease did not affect the ability of 'dead' S to transform R but DNAase did, therefore they concluded that the genetic material in transformation is most likely DNA.
Instead of using mice as Griffith did, the three scientists used a test tube assay. They found that this lysate changed R strains to S strains in the test tubes, so the component that caused the transformation had to be in the lysate. The tubes contained the protective sugar coating, proteins, DNA, and RNA.
After reaching this result, the scientists decided to separate the components and test each one individually. The heated S lysate was first introduced to the enzyme SIII, which destroyed the sugar coating (polysaccharides). When tested, they found that the sugar coat-less lysate still completed transformation. This meant that the R strain wasn't just using old pieces of the coating to assemble new coats. Following this, the scientists took the uncoated lysate and added protein-digesting enzymes called trypsin and chymotrypsin. This removed all proteins from the lysate. They tested this new product and found that it could still transform. It ruined the theory that proteins were the transforming component of the strains. The scientists' next step was to precipitate (make solid) DNA and RNA by using alcohol. They dissolved these in water and once again tested the lysate for transformation. RNA was destroyed using the RNase enzyme (this enzyme catalyzes the cleavage of RNA), and the remaining solution (no coat, no proteins, and no RNA) was tested for transformation. It was still able to transform, so RNA was not the factor. DNase was the last enzyme used to destroy DNA, the remaining component in the lysate. The scientists found that the product could not transform. Avery, MacLeod and McCarty determined that DNA was the transforming principle.