Method for preparing single-stranded dna
AssigneesKabushiki Kaisha Dnaform, Riken
InventorsAlexander Lezhava, Yuko Shibata, Matthias Harbers, Toshizo Hayashi, Yoshihide Hayashizaki, Piero Carninci
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Abstract
The invention is a method and a kit for preparing single-stranded DNA from double-stranded DNA and the purification of single-stranded DNA derived from double-stranded DNA. A single-stranded-DNA binding substance is used in combination with a double-strand-specific endonuclease for the removal of undesired double-stranded DNA from a single-stranded DNA preparation and for other related purposes.
Claims37
A method for purifying a single-stranded DNA from a mixture of the single-stranded DNA and a partially or entirely double-stranded DNA and/or DNA-RNA hybrid, comprising the steps of:
- having a single-stranded-DNA binding substance which binds to single-stranded DNA molecules attached to the single-stranded DNA molecules so as to protect them,
- digesting double-stranded DNA molecules by means of a double-strand-specific endonuclease which specifically cleaves double-stranded DNA and/or DNA-RNA molecules and which does not cleave single-stranded DNA to produce a digestion product, and
- separating the protected single-stranded DNA molecules from the digestion product.
A method for preparing a circular single-stranded DNA from a circular double-stranded DNA or a mixture of circular single- and double-stranded DNAs comprising the steps of:
- cutting a circular double-stranded DNA with an enzyme that has nicking activity to introduce a cut into one of the two DNA strands making up the circular double-stranded DNA,
- digesting the one strand cut by the nicking enzyme with an exonuclease to produce circular single-stranded DNA molecules,
- having a single-stranded-DNA binding substance which specifically binds to single-stranded DNA molecules attached to the single stranded DNA molecules so as to protect them,
- digesting remaining double-stranded DNA molecules by means of a double-strand-specific endonuclease which specifically cleaves double-stranded DNA molecules, and
- removing linear single-stranded DNA molecules from circular single-stranded DNA molecules by means of an exonuclease.
A method for removing double-stranded DNA molecules from a mixture of double-stranded and single-stranded DNA molecules, comprising the steps of:
- adding to the mixture a single-stranded-DNA binding substance which binds to single-stranded DNA molecules, and
- adding to the mixture a double-strand-specific endonulease which specifically cleaves double-stranded DNA molecules.
A method for removing nucleic acid molecules having a certain nucleic acid sequence and belonging to a tester, comprising the steps of:
- preparing driver oligonuleotide molecules that can hybridize to the nucleic acid molecules having the certain nucleic acid sequence and belonging to the tester,
- adding the driver oligonuleotide molecules in an excess amount compared to the amount of the nucleic acid molecules having the certain nucleic acid sequence to a sample containing the tester, and
- adding a double-strand-specific endonulease to the sample so as to digest double-stranded molecules formed between the driver oligonucleotide molecules and the nucleic acid molecules having the certain nucleic acid sequence.
A kit for purifying a single-stranded DNA from a mixture of the single-stranded DNA and a partially or entirely double-stranded DNA and/or DNA-RNA hybrid, comprising:
- a single-stranded-DNA binding substance which binds to single-stranded DNA molecules so as to protect them, and
- a double-strand-specific endonuclease which specifically cleaves double-stranded DNA molecules and which does not cleave single-stranded DNA to produce a digestion product.
A kit for preparing a circular single-stranded DNA from a circular double-stranded DNA or a mixture of circular single- and double-stranded DNAs comprising:
- an enzyme that has nicking activity to introduce a cut into one of the two DNA strands making up the circular double-stranded DNA,
- an exonuclease that digests the nicked strand for producing circular single-stranded DNA molecules,
- a single-stranded-DNA binding substance which binds to single-stranded DNA molecules so as to protect them, and
- a double-strand-specific endonuclease which specifically cleaves double-stranded DNA molecules and which does not cleave single-stranded DNA to produce a digestion product.
Dependencies are inferred from the claim text.