Are there any methods for nucleotide labeling which are compatible with use for reverse-transcription, besides radiolabeling, and for which the regulatory framework in the EU / USA are permissive ? Regulatory obligations around radionucleides being too strict.
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2 Answers
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Biotin-labeled nucleotides can be incorporated into cDNA by reverse transcriptase. Jena Bioscience, a provider of biotin-labeled nucleotides, has a table showing which nucleotide chemistries have been successfully used with reverse transcriptase, though you'll have to delve into the data sheets of specific products to get the references.
One reference provided on the product page for biotin-11-dCTP:
APOBEC3G Inhibits Elongation of HIV-1 Reverse Transcripts
This study looks at natural endogenous reverse transcription (nERT) in HIV. From the methods:
For this assay, a nERT reaction was performed essentially as above, with the exception of the dNTP addition: pelletted virions were resuspended in PBS, 2.5 mM MgCl2, 15 µg/ml melittin and 0.5mM biotin-11-dCTP (Jena Bioscience), and incubated at 37°C.
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Yes, Biotin is non radioactive option used for labeling of nucleotides. Various enzymes used for incorporating this biotin labelled nucleotide are: Taq Polymerase for Ploymerase Chain Reaction, DNA Polymerase for Nick Translation, Moloney Murine Leukemia Virus Reverse Transcriptase for Reverse Transcription, Klenow Exonuclease for Primer Extension and Terminal deoxynucleotidyl Transferase for 3'-end labeling.
The detection of these Biotin labeled probes is done using streptavidin conjugated with a horseradish peroxidase, alkaline phosphatase, or agarose/magnetic beads.
https://www.promocell.com/f/product-information/manual/PK-CA-BIO-DUTP-1000P.pdf)
