Health Information

In this Pulse Field Gel Electrophoresis technique, electric field is applied alternatively at definite period of time pulse (60 sec pulse).

Principle

DNA molecules are very long molecules. When electric field is applied these molecules stretch out to become a linear molecule before they migrate to the gel. When the direction is changed the smaller molecules realign and move faster compared to longer ones. Separation of long strands of DNA based on their charge by mass ratio on an agarose gel in the presence of alternating electric fields. This technique depends on differential stretching and relaxing of a large DNA molecule.

Apparatus

It consists of two or more electrodes arrayed around the periphery of agarose gel. Different electrode pairs are sequentially pulsed for varying times depending on the DNA being separated.

Procedure

This separation of DNA is carried on agarose gel. When electric field is applied in one direction for a fixed duration of time after which the direction of electric field is reversed. Smaller molecules reorient faster and migrate at a faster rate. Time required reorienting very long gel embedded DNA molecule increases with their size. Therefore choice of electrode distribution and pulse length causes shorter DNA to migrate through the gel faster than longer DNA. This process is done continuously and DNA fragments get separated effectively. The DNA that has been separated can be seen using Autoradiography and using ethidium bromide.

Application

Used for extensive study of human genome and for studying genome of yeast that contains 3-10 million base pairs. Whole chromosome is separated by electrophoresis.