Nuclease protection assay
Encyclopedia
Nuclease protection assay is a laboratory technique used in biochemistry
and genetics
to identify individual RNA
molecules in a heterogeneous RNA sample extracted from cells
. The technique can identify one or more RNA molecules of known sequence even at low total concentration
. The extracted RNA is first mixed with antisense RNA or DNA
probes that are complementary to the sequence or sequences of interest and the complementary strands are hybridized to form double-stranded RNA (or a DNA-RNA hybrid). The mixture is then exposed to ribonuclease
s that specifically cleave only single-stranded RNA but have no activity against double-stranded RNA. When the reaction runs to completion, susceptible RNA regions are degraded to very short oligomer
s or to individual nucleotide
s; the surviving RNA fragments are those that were complementary to the added antisense strand and thus contained the sequence of interest.
is used; when the probe is RNA, any single-strand-specific ribonuclease can be used. Thus the surviving probe-mRNA complement is simply detected by autoradiography.
s and 5' and 3' ends of transcribed gene
regions. Quantitative
results can be obtained regarding the amount of the target RNA present in the original cellular extract - if the target is a messenger RNA
, this can indicate the level of transcription
of the gene in the cell.
They are also used to detect the presence of double stranded RNA, presence of which could mean RNA interference.
Northern blot
ting, a laboratory technique that produces similar information but is slower and less quantitative, however, produces an accurate information about the size of the target RNA. Nuclease protection assay products are limited to the size of the initial probes due to the destruction of the non-hybridized RNA during the nuclease digestion step.
Biochemistry
Biochemistry, sometimes called biological chemistry, is the study of chemical processes in living organisms, including, but not limited to, living matter. Biochemistry governs all living organisms and living processes...
and genetics
Genetics
Genetics , a discipline of biology, is the science of genes, heredity, and variation in living organisms....
to identify individual RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
molecules in a heterogeneous RNA sample extracted from cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
. The technique can identify one or more RNA molecules of known sequence even at low total concentration
Concentration
In chemistry, concentration is defined as the abundance of a constituent divided by the total volume of a mixture. Four types can be distinguished: mass concentration, molar concentration, number concentration, and volume concentration...
. The extracted RNA is first mixed with antisense RNA or 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...
probes that are complementary to the sequence or sequences of interest and the complementary strands are hybridized to form double-stranded RNA (or a DNA-RNA hybrid). The mixture is then exposed to ribonuclease
Ribonuclease
Ribonuclease is a type of nuclease that catalyzes the degradation of RNA into smaller components. Ribonucleases can be divided into endoribonucleases and exoribonucleases, and comprise several sub-classes within the EC 2.7 and 3.1 classes of enzymes.-Function:All organisms studied contain...
s that specifically cleave only single-stranded RNA but have no activity against double-stranded RNA. When the reaction runs to completion, susceptible RNA regions are degraded to very short oligomer
Oligomer
In chemistry, an oligomer is a molecule that consists of a few monomer units , in contrast to a polymer that, at least in principle, consists of an unlimited number of monomers. Dimers, trimers, and tetramers are oligomers. Many oils are oligomeric, such as liquid paraffin...
s or to individual 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...
s; the surviving RNA fragments are those that were complementary to the added antisense strand and thus contained the sequence of interest.
Probe
The probes are prepared by cloning part of the gene of interest in a vector under the control of any of the following promoters, SP6, T7 or T3. These promoters are recognized by DNA dependent RNA polymerases originally characterized from bacteriophages. The probes produced are radioactive as they are prepared by in vitro transcription using radioactive UTPs. Uncomplemented DNA or RNA is cleaved off by nucleases. When the probe is a DNA molecule, S1 nucleaseS1 nuclease
S1 nuclease is an endonuclease that is active against single-stranded DNA and RNA molecules. It is five times more active on DNA than RNA. Its reaction products are oligonucleotides or single nucleotides with 5' phosphoryl groups...
is used; when the probe is RNA, any single-strand-specific ribonuclease can be used. Thus the surviving probe-mRNA complement is simply detected by autoradiography.
Uses
Nuclease protection assays are used to map intronIntron
An intron is any nucleotide sequence within a gene that is removed by RNA splicing to generate the final mature RNA product of a gene. The term intron refers to both the DNA sequence within a gene, and the corresponding sequence in RNA transcripts. Sequences that are joined together in the final...
s and 5' and 3' ends of transcribed gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
regions. Quantitative
Quantitative property
A quantitative property is one that exists in a range of magnitudes, and can therefore be measured with a number. Measurements of any particular quantitative property are expressed as a specific quantity, referred to as a unit, multiplied by a number. Examples of physical quantities are distance,...
results can be obtained regarding the amount of the target RNA present in the original cellular extract - if the target is a messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
, this can indicate the level of transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
of the gene in the cell.
They are also used to detect the presence of double stranded RNA, presence of which could mean RNA interference.
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,...
ting, a laboratory technique that produces similar information but is slower and less quantitative, however, produces an accurate information about the size of the target RNA. Nuclease protection assay products are limited to the size of the initial probes due to the destruction of the non-hybridized RNA during the nuclease digestion step.