Isopycnic centrifugation
Encyclopedia
Isopycnic
centrifugation, also known as density gradient centrifugation or equilibrium sedimentation is a technique used to separate molecules on the basis of buoyant density. (The word "isopycnic" means "equal density".) Typically, a "self-generating" density gradient is established via equilibrium sedimentation, and then analyte molecules concentrate as bands where the molecule density matches that of the surrounding solution. To illustrate the process, consider the fractionation of nucleic acids such as DNA
. To begin the analysis, a mixture of caesium chloride
and DNA is placed in a centrifuge
for several hours at high speed to generate a force of about 10^5 x g
(earth's gravity). Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA. After some time a gradient of the caesium ions is formed, caused by two opposing forces: diffusion
and centrifugal force. The sedimenting particles (caesium ions) will sediment away from the rotor, and become more concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of solvated caesium chloride and is always directed towards the center of the rotor. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is more dense near the bottom of the tube, and less dense near the top.
The DNA molecules will then be separated based on the relative proportions of AT (adenine
and thymine
base pairs) to GC (guanine
and cytosine
base pairs). An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than supercoiled
plasmid
DNA, which is denser than linear chromosomal DNA.
Isopycnic
Isopycnic means "of the same density." In particular, an isopycnic surface is a surface of constant density. This term is a bit more obscure than the similar terms isobaric or isothermal surfaces, which describe surfaces of constant pressure and constant temperature respectively...
centrifugation, also known as density gradient centrifugation or equilibrium sedimentation is a technique used to separate molecules on the basis of buoyant density. (The word "isopycnic" means "equal density".) Typically, a "self-generating" density gradient is established via equilibrium sedimentation, and then analyte molecules concentrate as bands where the molecule density matches that of the surrounding solution. To illustrate the process, consider the fractionation of nucleic acids such as DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
. To begin the analysis, a mixture of caesium chloride
Caesium chloride
Caesium chloride is the inorganic compound with the formula CsCl. This colorless solid is an important source of caesium ions in a variety of applications. Its crystal structure forms a major structural type where each caesium ion is coordinated by 8 chlorine ions...
and DNA is placed in a centrifuge
Centrifuge
A centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...
for several hours at high speed to generate a force of about 10^5 x g
G-force
The g-force associated with an object is its acceleration relative to free-fall. This acceleration experienced by an object is due to the vector sum of non-gravitational forces acting on an object free to move. The accelerations that are not produced by gravity are termed proper accelerations, and...
(earth's gravity). Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA. After some time a gradient of the caesium ions is formed, caused by two opposing forces: diffusion
Diffusion
Molecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
and centrifugal force. The sedimenting particles (caesium ions) will sediment away from the rotor, and become more concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of solvated caesium chloride and is always directed towards the center of the rotor. The balance between these two forces generates a stable density gradient in the caesium chloride solution, which is more dense near the bottom of the tube, and less dense near the top.
The DNA molecules will then be separated based on the relative proportions of AT (adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
and thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
base pairs) to GC (guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
and cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
base pairs). An AT base pair has a lower molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with the greater proportion of AT base pairs will have a lower density, all other factors being equal. Different types of nucleic acids will also be separated into bands, e.g. RNA is denser than supercoiled
DNA supercoil
DNA supercoiling refers to the over- or under-winding of a DNA strand, and is an expression of the strain on the polymer. Supercoiling is important in a number of biological processes, such as compacting DNA. Additionally, certain enzymes such as topoisomerases are able to change DNA topology to...
plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
DNA, which is denser than linear chromosomal DNA.