This research focuses on the preservation of the biological characteristics in the DNA splicing process by using Yusof-Goode (Y-G) splicing system. A splicing language results from a splicing system, which can be categorised into adult/inert persistent, transient, limit and active persistent language. Previously, only those types of splicing language are considered.  In this research, the concept of limit language has been extended to second order limit language.  A new notion of the second order limit language is introduced. Besides that, some restrictions on the Y-G splicing system that lead to the existence of non-second order limit language are discussed. Other than that, some sufficient conditions on the existence of the second order limit language in the Y-G splicing system are presented. In addition, a few biological examples are given to compare the difference between those languages. Some relations between second order limit language and various types of Y-G splicing system such as simple, semi-simple, semi-null, uniform splicing, Sk Hand null context splicing system are presented. Lastly, a laboratory experiment has been conducted to verify the mathematical model and also to validate the presence of second order limit language in the splicing system. DNA molecules that result from the experiment are the same as the predicted splicing language which show that the model is correct.