Patents Relating to Donor Vector

Patent or Publication No Title, Independent Claims and Summary Assignee
US 5888732

  • Earliest priority – 07 Jun 1995
  • Filed – 07 Jun 1996
  • Granted – 30 Mar 1999
  • Expected expiry – 30 Mar 2016 [Date of grant + 17 years?]
Title – Recombinational cloning using engineered recombination sites

Claim 1A Vector Donor DNA molecule comprising
a first DNA segment and a second DNA segment, said first or second DNA segment containing
at least one Selectable marker, wherein
i) said first or second DNA segment is flanked by at least a first and a second recombination site; and
ii) said first recombination site and said second recombination site do not recombine with each other.
Claim 8
An Insert Donor DNA molecule, comprising
a first DNA segment flanked by at least a first recombination site and a second recombination site, wherein
said first and second recombination sites do not recombine with each other.
Claim 12 
A kit comprising
at least one Vector Donor DNA molecule comprising
at least a first DNA segment and a second DNA segment, said first or second DNA segment containing at least one Selectable marker, wherein
said first or second DNA segment is flanked by at least a first and a second recombination site, that do not recombine with each other.
Claim 16A recombinant nucleic acid molecule, comprising
at least one DNA segment comprising
at least a first and a second recombination site flanking a Selectable marker or at least one desired DNA segmentwherein
at least one of said first and said second recombination sites comprises a core region that enhances recombination efficiency or specificity in vitro in the formation of a Cointegrate DNA or a Product DNA, and wherein
said first and second sites do not recombine with each other.
Claim 26 
A recombinant nucleic acid molecule, comprising
at least one recombination site comprising
at least one nucleic acid sequence having at least one of SEQ ID NOS:1-16, or a complementay DNA sequence or a corresponding RNA sequence.
Claim 29 
A method of making a Cointegrate DNA molecule, comprising combining in vitro.
(i) an Insert Donor DNA molecule, comprising
a desired DNA segment flanked by a first recombination site and a second recombination site, wherein
the first and second recombination sites do not recombine with each other;
(ii) a Vector Donor DNA molecule containing
a third recombination site and a fourth recombination site, wherein
the third and fourth recombination sites do not recombine with each other; and
(iii) at least one site specific recombination protein capable of recombining said first and third recombinational sites said second and fourth recombinational sites; thereby allowing recombination to occur, so as to produce a Cointegrate DNA molecule comprising
said first and third or said second and fourth recombination sites.
Claim 38 
A recombinant nucleic acid molecule comprising
at least a first and a second recombination site flanking at least one DNA segment containing
at least one Selectable marker, wherein
said first and second recombination sites do not recombine with each other.

The claims are generally drawn to:

  • A Vector Donor DNA molecule comprising a first and second DNA segment. The first or second DNA segment containing at least one Selectable marker, wherein
    • the first or second DNA segment is flanked by at least a first and a second recombination site; and
    • the first and second recombination sites do not recombine with each other.

This is potentially a very broad “product” claim, depending on the interpretation of the term “recombination site”.  In the broadest sense this might be read to imply a claim over vectors used in double Campbell recombination reactions.  Potentially it may cover all Gateway vectors, not just pDONR vectors. Is this claim justifiable? Is there overlap with prior art: such as vectors that have been used in Campbell recombination reactions?

  • An Insert Donor DNA molecule, comprising a first DNA segment flanked by at least a first and second recombination sites, wherein said first and second recombination sites do not recombine with each other.

Again, another potentially very broad product claim. This time not necessarily to a vector – it could also be interpreted to cover PCR products flanked by recombination sites.

  • A kit comprising at least one Vector Donor DNA molecule comprising at least a first and second DNA segment, said first or second DNA segment containing at least one Selectable marker, wherein said first or second DNA segment is flanked by at least a first and a second recombination site, that do not recombine with each other.

Claims a kit “comprising” a similar Vector Donor to that described above.

  • A recombinant nucleic acid molecule, comprising at least one DNA segment comprising at least a first and a second recombination site flanking a Selectable marker or at least one desired DNA segment, wherein at least one of the first and second recombination sites comprises a core region that enhances recombination efficiency or specificity in vitro in the formation of a Cointegrate DNA or a Product DNA, and wherein said first and second sites do not recombine with each other.

The term “do not recombine with each other” is a common feature of many of the patent claims.  This may be necessary to avoid issues of prior art. Theoretically, single-cross-over  Campbell recombination events can lead to integration and de-integration of two nucleic acid molecules. However, such DNA molecules (or vectors) mightcontain recombination sites that recombine with each other.

  • A recombinant nucleic acid molecule, comprising at least one recombination site comprising at least one nucleic acid sequence having at least one of SEQ ID NOS:1-16, or a complementay DNA sequence or a corresponding RNA sequence.

SEQ IDs 1-16 Refer to specific sequences found within the core recombination sites.  While SEQ IDs 6-16 are specific sequences found within the attB, attP, attR, and attL recombination sites, SEQ IDs 1-5 refer to sequences containing “wobbles”, and so claim many possible recombination sites. It is interesting to speculate that recombination sequences not listed within this group might constitute a work-around for this particular product claim.

  • A method of making a Cointegrate DNA molecule, comprising combining in vitro.
    • (i) an Insert Donor DNA molecule, comprising a desired DNA segment flanked by a first and a second recombination site, wherein the first and second recombination sites do not recombine with each other;
    • (ii) a Vector Donor DNA molecule containing a third and a fourth recombination site, wherein the third and fourth recombination sites do not recombine with each other; and
    • (iii) at least one site specific recombination protein capable of recombining said first and third recombinational sites and said second and fourth recombinational sites; thereby allowing recombination to occur, so as to produce a Cointegrate DNA molecule comprising the first and third or the second and fourth recombination sites.

Claims a method for creating the intermediate co-integrant DNA molecule.  Chimeric of both the Insert and Vector Donors.

  • A recombinant nucleic acid molecule comprising at least a first and a second recombination site flanking at least one DNA segment containing at least one Selectable marker, wherein said first and second recombination sites do not recombine with each other.
Life Technologies Inc.,
Rockville,MD.(Later acquired
by Invitrogen)
US6277608

  • Earliest priority – 23 Oct 1998
  • Filed – 22 Apr 1999
  • Granted – 06 Aug 2001
  • Expected expiry
Title –  Recombinational cloning using nucleic acids having recombination sites

Claim 1

A method for cloning or subcloning desired nucleic acid molecules comprising
a) combining in vitro or in vivo
i) one or more Insert Donor molecules comprising
one or more nucleic acid segments flanked by two or more recombination sites, wherein
said recombination sites do not substantially recombine with each other;
ii) two or more different Vector Donor molecules, each comprising
two or more recombination sites, wherein
said recombination sites do not substantially recombine with each other; and
iii) one or more site specific recombination proteins; and
b) incubating the combination under conditions sufficient to transfer one or more of said nucleic acid segments into said different Vector Donor molecules, thereby producing two or more different Product molecules.

The claims are generally drawn to:

  • A method for cloning or subcloning nucleic acid molecules comprising

    • a) combining in vitro or in vivo
      • i) one or more Insert Donor molecules comprising one or more nucleic acid segments flanked by two or more recombination sites, wherein said recombination sites do not substantially recombine with each other;
      • ii) two or more different Vector Donor molecules, each comprising two or more recombination sites, wherein said recombination sites do not substantially recombine with each other; and
      • iii) one or more site specific recombination proteins; and
    • b) incubating the combination under conditions sufficient to transfer one or more of said nucleic acid segments into said different Vector Donor molecules, thereby producing two or more different Product molecules.

Again, this is a broad methods claim. The language of the single independent claim of the ‘608 patent is different with respect to “substantially recombine with each other“, from “recombination sites do not recombine with each other” in the ‘732 patent above. What does “substantially” mean? Does this imply that some “low” level of recombination can occur between recombination sites? Further dependent claims specifiy the sequences of the recombination sites, as in the ‘732 patent above.