The toeprinting assay, also known as the primer extension inhibition assay, is a method used in

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

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 ...

and

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

RNA-binding protein RNA-binding proteins (often abbreviated as RBPs) are proteins that bind to the double or single stranded RNA in cell (biology), cells and participate in forming ribonucleoprotein complexes. RBPs contain various structural motifs, such as RNA reco ...

s. 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, free

nucleotides Nucleotides are Organic compound, organic molecules composed of a nitrogenous base, a pentose sugar and a phosphate. They serve as monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both o ...

, and

reverse transcriptase A reverse transcriptase (RT) is an enzyme used to convert RNA genome to DNA, a process termed reverse transcription. Reverse transcriptases are used by viruses such as HIV and hepatitis B to replicate their genomes, by retrotransposon mobi ...

(RT), among other

reagents In chemistry, a reagent ( ) or analytical reagent is a substance or compound added to a system to cause a chemical reaction, or test if one occurs. The terms ''reactant'' and ''reagent'' are often used interchangeably, but reactant specifies a ...

. The assay involves letting the RT generate

cDNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

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
Laboratory techniques Molecular biology techniques