Dot blot
Encyclopedia
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
used to detect biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
s. It represents a simplification of the northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
, Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
, or western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
methods. In a dot blot the biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
s to be detected are not first separated by chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
. Instead, a mixture containing the molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
to be detected is applied directly on a membrane as a dot. This is then followed by detection by either nucleotide
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
probes
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
(for a northern blot
Northern blot
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA in a sample. With northern blotting it is possible to observe cellular control over structure and function by determining the particular gene expression levels during differentiation,...
and Southern blot
Southern blot
A Southern blot is a method routinely used in molecular biology for detection of a specific DNA sequence in DNA samples. Southern blotting combines transfer of electrophoresis-separated DNA fragments to a filter membrane and subsequent fragment detection by probe hybridization. The method is named...
) or antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
(for a western blot
Western blot
The western blot is a widely used analytical technique used to detect specific proteins in the given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide...
).
The technique offers significant savings in time, as chromatography
Chromatography
Chromatography is the collective term for a set of laboratory techniques for the separation of mixtures....
or gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
, and the complex blotting procedures for the gel are not required. However, it offers no information on the size of the target biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
. Furthermore, if two molecule
Molecule
A molecule is an electrically neutral group of at least two atoms held together by covalent chemical bonds. Molecules are distinguished from ions by their electrical charge...
s of different sizes are detected, they will still appear as a single dot. Dot blots therefore can only confirm the presence or absence of a biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
or biomolecule
Biomolecule
A biomolecule is any molecule that is produced by a living organism, including large polymeric molecules such as proteins, polysaccharides, lipids, and nucleic acids as well as small molecules such as primary metabolites, secondary metabolites, and natural products...
s which can be detected by the DNA probes
Hybridization probe
In molecular biology, a hybridization probe is a fragment of DNA or RNA of variable length , which is used in DNA or RNA samples to detect the presence of nucleotide sequences that are complementary to the sequence in the probe...
or the antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
.
A radioactive sample can be hybridized to it allowing the researcher to detect variation between samples. The DNA is quantified and equal amounts are aliquoted into tubes in excess of the number of its targets in the samples, such as 10µg for a plasmid and 1µg for a PCR amplicon. These are denatured (NaOH and 95°C) and added to the wells where a vacuum sucks the water (with NaOH and NH4OAc) from underneath the membrane (nylon or nitrocellulose).