Method for cloning of a rare, specifically mutated cell

• US 20040096832A1
• Filed: 11/18/2002
• Published: 05/20/2004
• Est. Priority Date: 11/18/2002
• Status: Active Grant

• Current Assignee: PreGentis, Inc.
• Original Assignee: PreGentis, Inc.
• Inventors: Metz, Richard, Blaese, R. Michael, DiCola, Mike
• Granted Patent: US 6,929,917 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries

Abstract

The invention concerns a new method of detecting a rare product of a directed genetic alteration of a cultured cell. The method is applicable to any method of making the alteration provided that a pair of closely linked alterations can be made. The method consists of sequentially using allele specific polymerase chain reaction (PCR) to preferentially amplify sequences containing one of the two linked alterations coupled with a second method that detects the second change in the PCR product. The second method can be restriction digestion, traditional sequencing or pyro-sequencing. Experiments indicate that alterations as rare as one correctly altered copy in 10,000 cells can be detected.

Claims

1. In a method of making a specific directed mutation in the genome of a cell by a sequence-specific process, of the type wherein a rare cell in culture is mutated by the sequence-specific process and the rare mutated cell is cloned from the culture, the improvement comprising:

a) treating the culture with a sequence-specific process that introduces a 3′ and

a 5′

mutation in a genomic target, wherein the 3′ and

5′

mutations are separated by not more than 100 nucleotides;

b) forming replicate subcultures of the treated culture;

c) making a first polymerase chain reaction (PCR) product that contains the site of the 3′ and

5′

mutations using a sample of genomic DNA from a replicate of at least two subcultures of the treated culture;

d) making a second PCR product that contains the site of the 5′

mutation using the first PCR product, an AS-PCR primer that encodes to the 3′

nucleotide mutation and a forward primer that is not homologous to the site of the 5′

mutation;

e) identifying a positive subculture by the presence of the 5′

mutation in the second PCR product using template from the subculture;

f) subdividing a replicate of the positive subculture;

g) identifying a positive subdivision of the positive subculture by the presence of the 5′

mutation in cells of the subdivision; and

h) verifying that the positive subdivision contains a doubly substituted cell having both the 3′ and

5′

mutations and cloning the doubly substituted cell.

2. The method of claim 1, wherein step (g) is performed by reiteration of steps (b) through (f) with the modification that in each subsequent iteration of step (b) the subcultures are the subdivisions formed by the previous iteration of step (f).

3. The method of claim 2, wherein step (g) is performed at least twice by reiterations of modified steps (b) through (f).

4. The method of claim 1, wherein the 3′

terminal of the AS-PCR primer encodes the 3′

mutation.

5. The method of claim 1, wherein the presence of the 5′

mutation is identified by a restriction enzyme digest.

6. The method of claim 1, wherein the presence of the 5′

mutation is identified by pyrosequencing.

7. The method of claim 1, wherein the presence of the 5′

mutation is identified by a hybridization-dependent enzymatic degradation of an oligonucleotide probe that is labeled with a fluorescent dye and a quenching dye, wherein said degradation results in separation of the fluorescent and the quenching dye.

8. The method of claim 1, wherein the sequence-specific process is short fragment homologous recombination (SFHR).

9. The method of claim 8, wherein the SFHR is performed with an exogenous nucleic acid substantially free of the antisense strand.

10. The method of claim 8, wherein the SFHR is performed with an exogenous nucleic acid substantially free of the sense strand.

11. The method of claim 1, wherein the mutated cell is a yeast cell.

12. The method of claim 1, wherein the mutated cell is a mammalian cell.

13. The method of claim 1, wherein the mutated cell is a plant cell.

14. The method of claim 4, wherein the sequence-specific process is short fragment homologous recombination (SFHR).

15. The method of claim 14, wherein the SFHR is performed with an exogenous nucleic acid substantially free of the antisense strand.

16. The method of claim 14, wherein the SFHR is performed with an exogenous nucleic acid substantially free of the sense strand.

17. The method of claim 4, wherein the mutated cell is a yeast cell.

18. The method of claim 4, wherein the mutated cell is a mammalian cell.

19. The method of claim 4, wherein the mutated cell is a plant cell.