Method for cloning of a rare, specifically mutated cell

• US 6,929,917 B2
• Filed: 11/18/2002
• Issued: 08/16/2005
• Est. Priority Date: 11/18/2002
• Status: Expired due to Fees

• Current Assignee: PreGentis, Inc.
• Original Assignee: PreGentis, Inc.
• Inventors: Metz, Richard, Blaese, R. Michael, DiCola, Mike
• Primary Examiner(s): Leffers, Gerry
• Assistant Examiner(s): Akhavan, Ramin
• Application Number:
  US10/298,850
• Publication Number:
  US 20040096832A1
• Time in Patent Office: 1,002 Days
• Field of Search: 435/455, 435/471, 435/6, 435/69.1, 435/69.7, 435/71.1
• 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. A method of detecting and isolating a cell in a culture, which cell is mutated by a sequence-specific process, the method 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 allele-specific polymerase chain reaction (AS-PCR) primer that encodes the 3′

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,

wherein the sequence-specific process is short fragment homologous replacement (SFHR), the SFHR comprising the use of an exogenous nucleic acid having a sense strand, an antisense strand, or both.

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 exogenous nucleic acid is substantially free of the antisense strand.

9. The method of claim 1, wherein the exogenous nucleic acid is substantially free of the sense strand.

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

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

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

13. The method of claim 4, wherein the exogenous nucleic acid is substantially free of the antisense strand.

14. The method of claim 4, wherein the exogenous nucleic acid is substantially free of the sense strand.

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

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

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

18. The method of claim 1, wherein making a second PCR product comprises a PCR reaction conducted at a reduced level of hybridization stringency relative to hybridization stringencies that suppress the amplification of a first PCR template lacking the 3′

mutation.

19. The method of claim 18, wherein the second PCR product is produced in an amount that is not significantly affected by the presence or absence of the 3′

mutation.

20. The method of claim 18, wherein the PCR reaction is conducted at an annealing temperature that results in 500-fold to 1000-fold preferential amplification of a mutated template versus a nonmutated template.

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

and 5′

mutations are separated by between 1 and 50 nucleotides.

22. A method of detecting and isolating a cell in a culture, the cell having a 3′

and a 5′

mutation in a genomic target, the method comprising;

a) forming replicate subcultures of a culture comprising the cell;

b) 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 culture;

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

mutation using the first PCR product, an allele-specific polymerase chain reaction (AS-PCR) primer that encodes the 3′

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

mutation;

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

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

e) subdividing a replicate of the positive subculture;

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

mutation in cells of the subdivision; and

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

5′

mutations and cloning the doubly substituted cell.

23. The method of claim 22, wherein the 3′

and 5′

mutations are separated by not more than 100 nucleotides.

24. The method of claim 23, wherein the 3′

and 5′

mutations are separated by between 1 and 50 nucleotides.

25. The method of claim 22, wherein making a second PCR product comprises a PCR reaction conducted at a reduced level of hybridization stringency relative to hybridization stringencies that suppress the amplification of a first PCR template lacking the 3′

mutation.

26. The method of claim 25, wherein the second PCR product is produced in an amount that is not significantly affected by the presence or absence of the 3′

mutation.

27. The method of claim 25, wherein the PCR reaction is conducted at an annealing temperature that results in 500-fold to 1000-fold preferential amplification of a mutated template versus a nonmutated template.