Toeprinting assay

{{Short description|Method in molecular biology}}

{{more citations needed|date=March 2016}}

The toeprinting assay, also known as the primer extension inhibition assay,{{Cite book|title = Ribosomes|series = Methods in Enzymology|date = 1988-01-01|issn = 0076-6879|pmid = 2468068|pages = 419–425|volume = 164|first1 = D.|last1 = Hartz|first2 = D. S.|last2 = McPheeters|first3 = R.|last3 = Traut|first4 = L.|last4 = Gold| chapter=Extension inhibition analysis of translation initiation complexes |doi=10.1016/s0076-6879(88)64058-4| isbn=978-0-12-182065-7 }} is a method used in molecular biology that allows one to examine the interactions between messenger RNA and ribosomes or RNA-binding proteins.{{Cite journal|title = Quantitative analysis of ribosome–mRNA complexes at different translation stages|journal = Nucleic Acids Research|date = 2010-01-01|issn = 0305-1048|pmc = 2817456|pmid = 19910372|pages = e15|volume = 38|issue = 3|doi = 10.1093/nar/gkp1025|first1 = Nikolay E.|last1 = Shirokikh|first2 = Elena Z.|last2 = Alkalaeva|first3 = Konstantin S.|last3 = Vassilenko|first4 = Zhanna A.|last4 = Afonina|first5 = Olga M.|last5 = Alekhina|first6 = Lev L.|last6 = Kisselev|first7 = Alexander S.|last7 = Spirin}} 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.{{Cite journal|title = Inhibition of translation initiation on Escherichia coli gnd mRNA by formation of a long-range secondary structure involving the ribosome binding site and the internal complementary sequence.|journal = Journal of Bacteriology|date = 1995-11-01|issn = 0021-9193|pmc = 177509|pmid = 7592434|pages = 6560–6567|volume = 177|issue = 22|first1 = J T|last1 = Chang|first2 = C B|last2 = Green|first3 = R E|last3 = Wolf|doi = 10.1128/jb.177.22.6560-6567.1995}}

To do a toeprint assay, one needs the mRNA of interest, ribosomes, a DNA primer, free nucleotides, and reverse transcriptase (RT), among other reagents.{{Cite journal|title = Primer extension analysis of eukaryotic ribosome-mRNA complexes.|journal = Nucleic Acids Research|date = 1998-11-01|issn = 0305-1048|pmc = 147915|pmid = 9776744|pages = 4853–4859|volume = 26|issue = 21|first = M|last = Kozak|doi=10.1093/nar/26.21.4853}} The assay involves letting the RT generate cDNA until it gets blocked by any bound ribosomes, resulting in shorter fragments called toeprints when the results are observed on a sequencing gel.