Toeprinting assay

The toeprinting assay, also known as the primer extension inhibition assay,^[1] is a method used in molecular biology that allows one to examine the interactions between messenger RNA and ribosomes or RNA-binding proteins.^[2] 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.^[3]

To do a toeprint assay, one needs the mRNA of interest, ribosomes, a DNA primer, free nucleotides, and reverse transcriptase (RT), among other reagents.^[4] 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.

References

^ Hartz, D.; McPheeters, D. S.; Traut, R.; Gold, L. (1988-01-01). "Extension inhibition analysis of translation initiation complexes". Ribosomes. Methods in Enzymology. Vol. 164. pp. 419–425. doi:10.1016/s0076-6879(88)64058-4. ISBN 978-0-12-182065-7. ISSN 0076-6879. PMID 2468068.
^ Shirokikh, Nikolay E.; Alkalaeva, Elena Z.; Vassilenko, Konstantin S.; Afonina, Zhanna A.; Alekhina, Olga M.; Kisselev, Lev L.; Spirin, Alexander S. (2010-01-01). "Quantitative analysis of ribosome–mRNA complexes at different translation stages". Nucleic Acids Research. 38 (3): e15. doi:10.1093/nar/gkp1025. ISSN 0305-1048. PMC 2817456. PMID 19910372.
^ Chang, J T; Green, C B; Wolf, R E (1995-11-01). "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 of Bacteriology. 177 (22): 6560–6567. doi:10.1128/jb.177.22.6560-6567.1995. ISSN 0021-9193. PMC 177509. PMID 7592434.
^ Kozak, M (1998-11-01). "Primer extension analysis of eukaryotic ribosome-mRNA complexes". Nucleic Acids Research. 26 (21): 4853–4859. doi:10.1093/nar/26.21.4853. ISSN 0305-1048. PMC 147915. PMID 9776744.

External links

(A) Schematic of the primer extension inhibition (toeprint) assay

[1] Hartz, D.; McPheeters, D. S.; Traut, R.; Gold, L. (1988-01-01). "Extension inhibition analysis of translation initiation complexes". Ribosomes. Methods in Enzymology. Vol. 164. pp. 419–425. doi:10.1016/s0076-6879(88)64058-4. ISBN 978-0-12-182065-7. ISSN 0076-6879. PMID 2468068.

[2] Shirokikh, Nikolay E.; Alkalaeva, Elena Z.; Vassilenko, Konstantin S.; Afonina, Zhanna A.; Alekhina, Olga M.; Kisselev, Lev L.; Spirin, Alexander S. (2010-01-01). "Quantitative analysis of ribosome–mRNA complexes at different translation stages". Nucleic Acids Research. 38 (3): e15. doi:10.1093/nar/gkp1025. ISSN 0305-1048. PMC 2817456. PMID 19910372.

[3] Chang, J T; Green, C B; Wolf, R E (1995-11-01). "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 of Bacteriology. 177 (22): 6560–6567. doi:10.1128/jb.177.22.6560-6567.1995. ISSN 0021-9193. PMC 177509. PMID 7592434.

[4] Kozak, M (1998-11-01). "Primer extension analysis of eukaryotic ribosome-mRNA complexes". Nucleic Acids Research. 26 (21): 4853–4859. doi:10.1093/nar/26.21.4853. ISSN 0305-1048. PMC 147915. PMID 9776744.

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