Amplification and cloning of single DNA molecules using rolling circle amplification

• US 8,497,069 B2
• Filed: 05/01/2006
• Issued: 07/30/2013
• Est. Priority Date: 04/29/2005
• Status: Active Grant

• Current Assignee: Telesis Bio, Inc.
• Original Assignee: Synthetic Genomics, Inc.
• Inventors: Smith, Hamilton O., Hutchison, Clyde A. III
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US11/919,515
• Publication Number:
  US 20090176280A1
• Time in Patent Office: 2,647 Days
• Field of Search: 435/6, 435/91.2, 435/6.12
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2531/125 Rolling circle

Abstract

The present invention relates, e.g., to a method for amplifying a small number of copies (e.g. a single copy) of a single-stranded circular DNA molecule (e.g. having a size of about 5-6 kb) by an isothermal rolling circle mechanism, using random or partially random primers and a F29-type DNA polymerase. The method, which can also be used for amplifying DNAs by non-rolling types of multiple displacement amplification, comprises incubating the reaction components in a small volume, e.g. about 10 μl or less, such as about 0.6 μl or less. The degree of amplification can be about 109 fold, or higher. A method for cell-free cloning of DNA, using the rolling circle amplification method of the invention, is described.

Claims

1. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set; and

,

further wherein the random or partially random primers are resistant to exonuclease.

4. The method of claim 1, wherein the amplification is performed in a volume of 3 μ

l or less.

5. The method of claim 1, wherein the amplification is performed in a volume of 0.6 μ

l or less.

6. The method of claim 1, wherein the amplification is performed in a single reaction, and the DNA template is amplified by a factor of at least, or about 10⁹.

2. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

further wherein at least two nucleotides in each of the primers are linked by a phosphorothioate linkage.

3. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

further wherein the DNA template is a single copy.

7. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

wherein the ratio of polymerase to template is about 1.5×

10¹¹;

1 or less; and

,

further wherein the DNA template is amplified by multiple displacement amplification (MDA).

8. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement at an isothermal temperature,

wherein the amplification is performed

a) with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

further wherein the DNA template is a single-stranded circle, and the amplification is by rolling circle amplification (RCA).

9. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

further wherein the DNA polymerase is a φ

29-type polymerase or φ

29 polymerase.

10. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

a) with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and,

further wherein the method is performed in an automated nanomolar system.

11. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed

a) with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set, and

b) in a volume of 5 μ

l or less, wherein the random or partially random primers are resistant to exonuclease.

16. The method of claim 11, further wherein the amplification is performed in a single reaction, and the DNA template is amplified by a factor of at least, or about 10⁹.

17. The method of claim 11, further wherein the DNA template is amplified by multiple displacement amplification (MDA).

18. The method of claim 11, further wherein the DNA template is a single-stranded circle, and the amplification is by rolling circle amplification (RCA).

19. The method of claim 11, further wherein the DNA polymerase is a φ

29-type polymerase or φ

29 polymerase.

20. The method of claim 11, further wherein the method is performed in an automated nanomolar system.

12. A method for amplifying a DNA template, comprising contacting a DNA with a strand-displacing, processive DNA polymerase and a set of random or partially random primers, under conditions that are effective for promoting DNA strand displacement, at an isothermal temperature,

wherein the amplification is performed with a primer set in which the primers cannot base pair their 3′

nucleotides with other copies of the primers, wherein if the 3′

terminal nucleotide of the primer set is A, the nucleotide T is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is T, the nucleotide A is not present at any position in the primer set;

if the 3′

terminal nucleotide of the primer set is C, the nucleotide G is not present at any position in the primer set; and

if the 3′

terminal nucleotide of the primer set is G, the nucleotide C is not present at any position in the primer set.

13. The method of claim 12, further wherein the random or partially random primers are resistant to exonuclease.

14. The method of claim 12, further wherein at least two nucleotides in each of the primers are linked by a phosphorothioate linkage.

15. The method of claim 12, further wherein the DNA template is a single copy.

21. The method of claim 12, further wherein the amplification is performed in a single reaction, and the DNA template is amplified by a factor of at least, or about 10⁹.

22. The method of claim 12, further wherein the DNA template is amplified by multiple displacement amplification (MDA).

23. The method of claim 12, further wherein the DNA template is a single-stranded circle, and the amplification is by rolling circle amplification (RCA).

24. The method of claim 12, further wherein the DNA polymerase is a φ

29-type polymerase or φ

29 polymerase.

25. The method of claim 12, further wherein the method is performed in an automated nanomolar system.