Unrestricted mutagenesis and cloning methods

A method of making an insertion in a nucleic acid comprising the steps of:

• contacting an Original DNA, which comprises a Target DNA Fragment containing a Modification Target that is flanked by two restriction sites with a DNA polymerase and two Starter Primers, the Starter Primers selected to amplify the Target DNA Fragment, so that the Starter Primers anneal to segments of the Original DNA at the ends of the Target DNA Fragment;
• amplifying the Target DNA Fragment using polymerase chain reaction (PCR) with the Starter Primers to generate a Starter DNA;
• self-ligating the Starter DNA with a DNA ligase to form a Closed Starter DNA;
• contacting the Closed Starter DNA with a DNA polymerase and two Opener Primers, the Opener Primers selected to insert at least one unique restriction site into the Modification Target, at least one of the Opener Primers comprising a 5′ extension including at least one additional unique restriction site that differs from the restriction sites in the Target DNA Fragment, so that the Opener Primers anneal to opposite strands of the adjacent segments of the Target DNA Fragment;
• amplifying the Closed Starter DNA using PCR with the Opener Primers to generate an Intermediate DNA;
• amplifying a DNA Insert with primers comprising the unique restriction sites that differ from the restriction sites in the Target DNA Fragment;
• digesting the Intermediate DNA and the DNA Insert with restriction enzymes corresponding to the unique restriction sites that were added;
• purifying a fragment of the Intermediate DNA and a fragment of the DNA Insert;
• ligating the purified fragments of the Intermediate DNA and of the DNA Insert to generate Closed Intermediate DNA;
• and, optionally,
• contacting the Closed Intermediate DNA with the Starter Primers and a DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA.

A method of deleting a portion of a nucleic acid comprising the steps of:

• contacting an Original DNA, comprising a Target DNA Fragment containing a Modification Target that is flanked by two restriction sites, with'a DNA polymerase and two Starter Primers that are selected to amplify the Target DNA Fragment, so that the Starter Primers anneal to segments of the Original DNA outside the restriction sites;
• amplifying a portion of the Original DNA comprising the Target DNA Fragment using polymerase chain reaction (PCR) with the Starter Primers to generate a Starter DNA;
• self-ligating the Starter DNA with a DNA ligase to form a Closed Starter DNA;
• contacting the Closed Starter DNA with two Opener Primers and a DNA polymerase, so that the Opener Primers anneal to nonadjacent segments of the DNA flanking the intended deletion and to different DNA strands;
• amplifying the Starter DNA by PCR with the Opener Primers to generate an Intermediate DNA;
• self-ligating the Intermediate DNA with DNA ligase to generate a Closed Intermediate DNA;
• and, optionally,
• contacting the Closed Intermediate DNA with the Starter Primers and DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA containing the deletion.

A method of modifying a nucleic acid comprising the steps of:

• contacting an Original DNA, comprising a Target DNA Fragment containing a Modification Target that is flanked by two restriction sites, with a DNA polymerase and two OIL Starter Primers, selected to amplify the target DNA Fragment, so that the OIL Starter Primers anneal to segments of the Original DNA at the ends of the Target DNA Fragment;
• amplifying a portion of the Original DNA comprising the Target DNA fragment using polymerase chain reaction (PCR) with the OIL Starter Primers to generate a Starter DNA;
• contacting the Starter DNA with a DNA polymerase and two Opener Primers, selected to generate a modification in the Target DNA Fragment, so that the Opener Primers anneal to opposite strands of the adjacent segments of the Target DNA Fragment;
• amplifying the Starter DNA using PCR with the OIL Starter Primers and the Opener Primers to generate two PCR products whose 3′ ends anneal and act as primers to complete the Intermediate DNA;
• self-ligating the Intermediate DNA with DNA ligase to generate a Closed Intermediate DNA;
• and, optionally,
• contacting the Closed Intermediate DNA with the OIL Primers and a DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA containing the modification.

A method of modifying a nucleic acid comprising the steps of:

• contacting in a first reaction mixture an Original DNA, comprising a Target DNA Fragment containing a Modification Target flanked by restriction sites, with OIL Starter Primer A and Opener Primer B;
• amplifying a portion of the Original DNA comprising the Target DNA fragment in the first reaction mixture to generate a portion of a first Intermediate DNA;
• contacting in a second reaction mixture the Original DNA with OIL Starter Primer B and Opener Primer A;
• amplifying a portion of the Original DNA comprising the Target DNA fragment in the second reaction mixture to generate a portion of a second Intermediate DNA;
• combining and extending the first and second Intermediate DNAs to generate a third Intermediate DNA, which may be optionally amplified with Opener Primers A and B;
• self-ligating the third Intermediate DNA with DNA ligase to generate a Closed Intermediate DNA;
• and, optionally,
• contacting the Closed Intermediate DNA with the OIL Primers and a DNA polymerase to amplify the Closed Intermediate DNA and generate a Linear Modified DNA containing the modification, wherein the OIL Primers anneal to segments of the Original DNA at the ends of the Target DNA Fragment and wherein the OIL Primers are designed so that the Target DNA Fragment is amplified.