How Is Recombinant DNA Identified?

rDNA is DNA that is made from two separate strands of DNA.

All the information needed to create an organism resides in its DNA. A base consisting of sugar, phosphate and one nitrogen base makes up DNA. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C). Each nitrogen base always pairs with its counterpart. Adenine will pair with thymine and cytosine will pair with guanine. All organisms carry the same four nitrogen bases. What separates one organism from another is the number of base pairings, and the sequence of their arrangements. Protein gives an organism its structure. A complex chemical process turns DNA into messenger RNA (mRNA) and mRNA eventually becomes translated into protein, which gives the organism its ultimate structure.

rDNA Definition

Recombinant DNA (rDNA) is the general name for taking a piece of one DNA, and combining it with another strand of DNA. It helps if you think about rDNA as chimera DNA. (A chimera was an ancient Greek monster consisting of a lion's body with a goat's head coming out of the spine with a snake for the tail.) When scientists combine two or more different strands of DNA, they are able to create a new strand of DNA. Combining the DNA of two different organisms is the most common recombinant process.

rDNA Production

Before you can understand how to identify rDNA, you must have an understanding of how scientists create rDNA in the first place. The three methods of rDNA production are transformation, non-bacterial transformation and phage introduction.

Transformation

In transformation, you insert a selected piece of DNA into a vector, and then you cut that piece of DNA with a restriction enzyme. Then you use DNA ligase to place the DNA insert into the vector. The vector is inserted into a host cell (usually E. coli), which has been specially prepared to take up the foreign DNA.

Non-bacterial Transformation

This is similar to transformation, with the exception that the host cells are non-bacterial. Either you insert the vector through microinjection where the DNA is inserted directly into the nucleus, or through biolistics where high velocity micro projectiles bombard the host cells until the vector DNA is absorbed.

Phage Introduction

Phage introduction is the process of transfection, which is equivalent to transformation. Instead of using a bacterium, you would use a phage such as lambda or M13.

rDNA Identification

When you are preparing your vector, you will format them with a selectable marker. This marker will enable you and others to distinguish transformed hosts with rDNA from untransformed hosts. The most often used selectable marker is an antibiotic marker. By using an antibiotic marker, you can quickly identify which cells contain rDNA because exposure to certain antibiotics will kill a host cell without rDNA because it is not resistant to the antibiotic. Any cells that are resistant to the antibiotic are those that contain the vector and, therefore, the rDNA.

References

• RDI: An Introduction to Recombinate DNA