Targeted sequence addition
AssigneeKeygene N.V.
InventorsRené Cornelis Josephus HOGERS, Stefan John WHITE, Theodorus Frank Maria ROELOFS
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Abstract
The invention pertains to a method for labelling a target nucleic acid fragment using a combination of a site-specific nuclease and a reverse transcriptase. The labelling results in the addition of a specific nucleotide sequence to at least one free 3′-end of the target nucleic acid fragment. The invention further relates to a method for determining the sequence of the target nucleic acid fragment as well as construct and kit for use in the method of the invention.
Claims18
A method for labelling a target nucleic acid fragment, wherein the target nucleic acid fragment comprises a first strand and a complementary second strand and wherein the target nucleic acid fragment comprises a sequence of interest, wherein the method comprises the steps of:
- a) providing a sample comprising a double-stranded nucleic acid molecule, wherein the double-stranded nucleic acid molecule comprises the sequence of interest;
- b) contacting the double-stranded nucleic acid molecule with a site-specific nuclease to generate a double-stranded break, wherein the double-stranded break results in a free 3′-end of the first strand of the target nucleic acid fragment;
- and
- c) contacting the cleaved nucleic acid molecule with a DNA polymerase and a template molecule, preferably contacting the cleaved nucleic acid molecule with a reverse transcriptase and a template RNA molecule, thereby labelling the free 3′-end of the first strand of the target nucleic acid fragment with one or more nucleotides,
- wherein optionally the site-specific nuclease in step b) and the reverse transcriptase in step c) are separate entities.
Dependencies are inferred from the claim text.