How is Recombinant DNA made?

There are three different methods by which Recombinant DNA is made. 

1. Transformation

The first step in transformation is to select a piece of DNA to be inserted into a vector. The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase. The insert contains a selectable marker which allows for identification of recombinant molecules. An antibiotic marker is often used so a host cell without a vector dies when exposed to a certain antibiotic, and the host with the vector will live because it is resistant.

The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA.

Selectable markers can be for antibiotic resistance, color changes, or any other characteristic which can distinguish transformed hosts from untransformed hosts.

2. Non-Bacterial Transformation

This is a process very similar to Transformation, which was described above. The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.

In microinjection, the DNA is injected directly into the nucleus of the cell being transformed. In biolistics, the host cells are bombarded with high velocity microprojectiles, such as particles of gold or tungsten that have been coated with DNA.

3. Phage Introduction

Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packagings of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.