Method of inserting unique DNA sequences into DNA vectors

• US 4,528,266 A
• Filed: 09/30/1982
• Issued: 07/09/1985
• Est. Priority Date: 10/01/1979
• Status: Expired due to Term

• Current Assignee: George Pieczenik
• Original Assignee: George Pieczenik
• Inventors: Pieczenik, George
• Primary Examiner(s): Tanenholtz, Alvin E.
• Application Number:
  US06/428,538
• Time in Patent Office: 1,013 Days
• Field of Search: 435/6, 435/91, 435/172, 435/317, 935/29, 935/77
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids

Abstract

Autonomously replicating DNA containing a unique nucleotide sequence, being an oligonucleotide of which its sequence does not otherwise exist in said DNA[inserted in a non-essential region thereof at a site not previously susceptible of restriction endonuclease cleavage].

Claims

1. A method of inserting a unique oligonucleotide sequence into a non-essential region of an autonomously replicating DNA element, comprising,

isolating double stranded circular molecules of the DNA, each having the same nucleotide sequence,

cleaving the circular DNA at random with respect to nucleotide sequence, producing a population of linear, double stranded DNA molecules comprising circular permutations of the same nucleotide sequence,

joining a unique oligonucleotide sequence to the ends of the linear DNA molecules, said unique oligonucleotide sequence not otherwise existing in said DNA element, then

rejoining the ends to form circular double stranded DNA molecules having the oligonucleotide of unique sequence inserted at random with respect to the nucleotide sequence of each circular DNA molecule,

transferring the circular DNA having said unique insert sequence to a host organism under conditions permitting replication of the DNA, and

selecting for progeny of the circular DNA having a unique insert sequence, said progeny bearing said insert in a non-essential region of the DNA.

4. The method of claim 1 wherein the unique oligonucleotide sequence is inserted into a structural gene of the autonomously replicating DNA element by providing growth conditions for a host organism such that the structural gene for receiving the insert is functionally non-essential, whereby the circular DNA having said unique sequence in said structural gene is transferred to said host organism under conditions permitting replication of the DNA.

2. A method of making a population of linear DNA molecules having the same nucleotide sequence circularly permuted and having a unique oligonucleotide sequence covalently attached at the ends thereof, said unique oligonucleotide sequence not otherwise existing in said DNA molecules comprising:

(a) isolating circular DNA of an autonomously replicating DNA element,

(b) cutting the circular DNA at random, thereby generating a population of linear DNA molecules having the same nucleotide sequence circularly permuted, and

(c) covalently attaching said unique oligonucleotide sequence to the ends of the linear DNA molecules of step (b).

3. A method of making a modified autonomously replicating DNA element wherein the modification comprises a unique oligonucleotide sequence not otherwise existing in said DNA element, said sequence inserted randomly with respect to the nucleotide sequence of said DNA element, comprising the steps of:

(a) isolating and purifying circular DNA of an autonomously replicating DNA element,

(b) cutting the circular DNA at random, thereby generating a population of linear DNA molecules having the same nucleotide sequence circularly permuted,

(c) covalently attaching said unique oligonucleotide sequence to the ends of the linear DNA molecules of step (b),

(d) covalently attaching the ends of the linear DNA molecules intramolecularly, whereby circular DNA containing said unique oligonucleotide sequence inserted randomly with respect to nucleotide sequence is made.

5. The method of genotypically mapping an automatically replicating DNA element comprising the steps of:

(a) providing a population of linear DNA molecules of said DNA element, comprising circular permutations of the same nucleotide sequence,

(b) attaching a unique oligonucleotide sequence to the ends of the linear DNA molecules of step (a), said unique sequence not otherwise existing in said DNA element,

(c) rejoining the ends of the linear molecules of step (b), thereby forming a population of circular autonomously replicating DNA molecules having said unique oligonucleotide sequence inserted randomly with respect to the nucleotide sequence,

(d) transforming or transfecting a population of host cells with the population of circular DNA molecules resulting from step (c),

(e) isolating clones of the DNA molecules of step (c), said clones having the unique oligonucleotide sequence inserted in a non-essential region of the autonomously replicating DNA element, under preselected growth conditions, and

(f) measuring the relative positions of the unique oligonucleotide sequence inserts of individual clones, with respect to the nucleotide sequence of the DNA element, thereby mapping the nonessential regions of its genome, under the preselected growth conditions, without reference to or knowledge of the function of any gene therein, said mapping being genotypic.