Method for the directional cloning of DNA
First Claim
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1. A method for directionally cloning an insert DNA sequence into a target DNA sequence comprising:
- generating a monophosphorylated target DNA sequence;
generating a monophosphorylated insert DNA sequence; and
combining said insert DNA sequence with said target sequence, wherein said insert sequence can ligate in only one orientation with respect to said target sequence.
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Abstract
A method for directionally cloning an insert DNA fragment into a target sequence using differential phosphorylation is disclosed, Monophosphorylated PCR fragments are directionally cloned into a monophosphorylated plasmid, Methods for directionally cloning non-PCR fragments into target DNA sequences are also discussed.
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Citations
16 Claims
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1. A method for directionally cloning an insert DNA sequence into a target DNA sequence comprising:
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generating a monophosphorylated target DNA sequence; generating a monophosphorylated insert DNA sequence; and combining said insert DNA sequence with said target sequence, wherein said insert sequence can ligate in only one orientation with respect to said target sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for directionally cloning a PCR generated DNA fragment into a target DNA comprising:
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generating a PCR fragment having only one phosphorylated 5'"'"' end; digesting a target DNA sequence with a blunt-end restriction enzyme; dephosphorylating the 5'"'"' ends of said target sequence; cleaving said target sequence to expose one 5'"'"' phosphate; and ligating said PCR generated DNA fragment with said target sequence, wherein said PCR generated DNA sequence can ligate in only one orientation with respect to said target sequence. - View Dependent Claims (9, 10, 11, 12, 13, 14, 15, 16)
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Specification