Colony Blots and Southern Blots

The heart of both a colony blot and a Southern blot is the use of a probe to locate a specific nucleotide sequence. The probe is a single-stranded piece of nucleic acid that has been tagged, or labeled, with a detectable marker such as a radioactive isotope, a fluorescent dye, or biotin; the probe is complementary to either of the two strands being detected. The DNA of the specimen being examined is affixed to a solid surface such as a nylon membrane and treated with an alkaline solution to denature it. Once the probe has been given enough time to hybridize, unbound probe is washed off. The presence and location of bound probe can be determined by assaying for the detectable marker.

Colony Blots

Colony blots are used to detect a given nucleotide sequence in a crude preparation of genomic DNA obtained from colonies grown on an agar plate. The procedure is done in such a way that a researcher can readily determine which colonies on the plate contain the sequence of interest (figure 9.7). The technique can be used to determine which cells in a collection of clones contain the DNA of interest; this process will be described later.

Southern Blots

Southern blots are used to detect a given sequence in restriction fragments that have been separated according to size and transferred to a membrane filter (figure 9.8). A technique called gel electrophoresis is used to separate the fragments. ■ gel electrophoresis, p. 236

The most obvious application of a Southern blot is to locate sequences that are homologous to the one being studied, which is how many functionally related genes have been discovered and characterized. This information can also be used to simplify a cloning experiment. For example if the gene of interest is shown to be encoded on a restriction fragment that is 6,000 base pairs in length, then fragments of that size can be separated from the rest and cloned.

Chapter 9 Biotechnology and Recombinant DNA

Colonies on agar plate

Colonies transferred

Chapter 9 Biotechnology and Recombinant DNA

Colonies transferred

Colonies on agar plate

— Nylon membrane

Membrane soaked in an alkaline solution

— Nylon membrane

Membrane soaked in an alkaline solution

Cells in colonies lyse and DNA is denatured

Was this article helpful?

0 0
You Are What You Eat

You Are What You Eat

Nutrition is a matter that people spend their careers learning about and requires volumes of books to explain. My objective is to instruct you how to consume a healthy nutritional diet that aids your body in burning off fat instead of storing it. You do not require overwhelming science to get this.

Get My Free Ebook


Post a comment