Nuclease protection assay is a laboratory technique used in

biochemistry Biochemistry or biological chemistry is the study of chemical processes within and relating to living organisms. A sub-discipline of both chemistry and biology, biochemistry may be divided into three fields: structural biology, enzymology and ...

and

genetics Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar wor ...

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 In chemistry, concentration is the abundance of a constituent divided by the total volume of a mixture. Several types of mathematical description can be distinguished: '' mass concentration'', ''molar concentration'', '' number concentration'', ...

. The extracted RNA is first mixed with

antisense In molecular biology and genetics, the sense of a nucleic acid molecule, particularly of a strand of DNA or RNA, refers to the nature of the roles of the strand and its complement in specifying a sequence of amino acids. Depending on the context ...

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 Ribonuclease (commonly abbreviated RNase) 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 ...

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 oligomers or to individual

nucleotide Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecule ...

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 nuclease Nuclease S1 () is an endonuclease enzyme that splits single-stranded DNA (ssDNA) and RNA into oligo- or mononucleotides. This enzyme catalyses the following chemical reaction : Endonucleolytic cleavage to 5'-phosphomononucleotide and 5'-phosph ...

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 introns and 5' and 3' ends of transcribed

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

regions.

Quantitative Quantitative may refer to: * Quantitative research, scientific investigation of quantitative properties * Quantitative analysis (disambiguation) * Quantitative verse, a metrical system in poetry * Statistics, also known as quantitative analysis ...

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 Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, the fir ...

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 The northern blot, or RNA blot,Gilbert, S. F. (2000) Developmental Biology, 6th Ed. Sunderland MA, Sinauer Associates. is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample ...

ting is a laboratory technique that produces similar information. It is slower and less quantitative, but also produces 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.

References

Sandelin, A. et al. Mammalian RNA polymerase II core promoters: insights from genome-wide studies. Nature Rev. Genet. 8, 424–436 (2007)
Biochemistry methods Genetics Molecular biology techniques {{biochem-stub