The toeprinting assay, also known as the primer extension inhibition assay, is a method used in molecular biology that allows one to examine the interactions between

messenger RNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of synthesizing a protein. mRNA is created during the p ...

and

ribosomes Ribosomes ( ) are macromolecular machines, found within all cells, that perform biological protein synthesis (mRNA translation). Ribosomes link amino acids together in the order specified by the codons of messenger RNA (mRNA) molecules to f ...

or RNA-binding proteins. It is different from the more commonly used DNA footprinting assay. The toeprinting assay has been utilized to examine the formation of the translation initiation complex. To do a toeprint assay, one needs the mRNA of interest, ribosomes, a DNA

primer Primer may refer to: Arts, entertainment, and media Films * ''Primer'' (film), a 2004 feature film written and directed by Shane Carruth * ''Primer'' (video), a documentary about the funk band Living Colour Literature * Primer (textbook), a t ...

, free nucleotides, and

reverse transcriptase A reverse transcriptase (RT) is an enzyme used to generate complementary DNA (cDNA) from an RNA template, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, ...

(RT), among other reagents. The assay involves letting the RT generate

cDNA In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...

until it gets blocked by any bound ribosomes, resulting in shorter fragments called toeprints when the results are observed on a sequencing gel.

References

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External links

(A) Schematic of the primer extension inhibition (toeprint) assay
Molecular biology Laboratory techniques Molecular biology techniques