COMPOSITIONS AND METHODS FOR NUCLEIC ACID AMPLIFICATION

• US 20160333340A1
• Filed: 05/10/2016
• Published: 11/17/2016
• Est. Priority Date: 05/11/2015
• Status: Abandoned Application

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: WU, Cheng-Hsien
• Application Number:
  US15/151,316
• Publication Number:
  US 20160333340A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important

Abstract

The present disclosure provides compositions, methods and systems for the efficient removal of a non-target nucleic acid sequence from a double stranded nucleic acid amplification product. A non-target nucleic acid sequence may be a primer sequence incorporated into the double stranded nucleic acid molecule during a nucleic acid synthesis or amplification reaction. The non-target nucleic acid sequence may include a nucleobases that are not canonical DNA nucleobases, such as uracil, that can be selectively removed as part of the non-target sequence removal process.

Claims

1. A method for nucleic acid amplification comprising:

a) providing a double stranded template nucleic acid comprising a first strand and a second strand;

b) mixing the double stranded template nucleic acid with a first primer and a second primer, wherein;

i) the first primer comprises a first plurality of nucleobases that are not canonical DNA nucleobases, wherein each nucleobase of the first plurality of nucleobases that are not canonical DNA nucleobases is distanced from another by about 3 to about 8 nucleobases, and wherein one nucleobase of the first plurality of nucleobases that are not canonical DNA nucleobases is a 3′

terminal nucleobase bound to a preceding nucleobase in the first primer by a phosphorothioate bond, and

ii) the second primer comprises a second plurality of nucleobases that are not canonical DNA nucleobases, wherein each nucleobase of the second plurality of nucleobases that are not canonical DNA nucleobases is distanced from another by about 3 to about 8 nucleobases, and wherein one nucleobase of the second plurality of nucleobases that are not canonical DNA nucleobases is a 3′

terminal nucleobase bound to a preceding nucleobase in the second primer by a phosphorothioate bond; and

c) amplifying the double stranded template nucleic acid by extending the first primer to form a first extension strand and extending the second primer to form a second extension strand; and

d) removing the first primer from the first extension strand and the second primer from the second extension strand.

2. The method of claim 1, wherein the first plurality of nucleobases that are not canonical DNA nucleobases or the second plurality of nucleobases that are not canonical DNA nucleobases comprises 3 or 4 nucleobases.

3. The method of claim 1, wherein each nucleobase of the first plurality of nucleobases that are not canonical DNA nucleobases is distanced from another by about 4 to about 7 nucleobases.

5. The method of claim 1, wherein the first primer and the second primer each have a total length of from about 12 to about 50 nucleobases.

6. The method of claim 1, wherein each nucleobase of the first plurality of nucleobases that are not canonical DNA nucleobases or each nucleobase of the second plurality of nucleobases that are not canonical DNA nucleobases is uracil, 3-meA (3-methyladenine), hypoxanthine, 8-oxoG (7,8-dihydro-8-oxoguanine), FapyG, FapyA, Tg (thymine glycol), hoU (hydroxyuracil), hmU (hydroxymethyluracil), fU (formyluracil), hoC (hydroxycytosine), fC (formylcytosine), 5-meC (5-methylcytosine), 6-meG (O6-methylguanine), 7-meG (N7-methylguanine), EC (ethenocytosine), 5-caC (5-carboxylcytosine), 2-hA, ε

A (ethenoadenine), 5-fU (5-fluorouracil), 3-meG (3-methylguanine), or isodialuric acid.

7. The method of claim 6, wherein the first plurality of nucleobases that are not canonical DNA nucleobases and the second plurality of nucleobases that are not canonical DNA nucleobases are the same type of nucleobase.

8. The method of claim 1, wherein each nucleobase of the first plurality of nucleobases that are not canonical DNA nucleobases and each nucleobase of the second plurality of nucleobases that are not canonical DNA nucleobases is uracil.

9. The method of claim 8, wherein the nucleobase preceding the 3′

terminal uracil nucleobase in the first primer is a pyrimidine, or wherein the nucleobase preceding the 3′

terminal uracil nucleobase in the second primer is a pyrimidine.

11. The method of claim 9, wherein the pyrimidine is a cytosine.

12. The method of claim 8, wherein the first primer is removed from the first extension strand with an efficiency of at least about 90%, and the second primer is removed from the second extension strand with an efficiency of at least about 90%.

13. The method of claim 1, wherein the melting temperature of the first primer or the second primer is between about 60°

C. and about 66°

C.

15. The method of claim 1, wherein the GC content of the first primer, the second primer, or both the first primer and the second primer is between about 40% and about 60%.

16. The method of claim 8, wherein removing the first primer from the first extension strand comprises excising the first plurality of uracil nucleobases from the first primer from the first extension strand, and wherein removing the second primer from the second extension strand comprises excising the second plurality of uracil nucleobases from the second primer from the second extension strand.

17. The method of claim 16, wherein excision of the first plurality of uracil nucleobases from the first primer of the first extension strand and excision of the second plurality of uracil nucleobases from the second primer of the second extension strand is achieved by treating the first extension strand and the second extension strand with a mixture of Uracil DNA glycosylase and a DNA glycosylase-lyase Endonuclease VIII.

18. The method of claim 8, wherein the first strand of the double stranded template nucleic acid comprises in 5′

to 3′

order;

a first primer binding site complementary to the first primer, and a target nucleic acid sequence; and

the second strand of the double stranded template nucleic acid comprises in 5′

to 3′

order;

a second primer binding site complementary to the second primer, and a nucleic acid sequence complementary to the target nucleic acid sequence.

19. The method of claim 18, wherein the first strand of the double stranded template nucleic acid comprises in 5′

to 3′

order;

the first primer binding site complementary to the first primer, the target nucleic acid sequence, and a nucleic acid sequence complementary to the second primer binding site; and

the second strand of the double stranded template nucleic acid comprises in 5′

to 3′

order;

the second primer binding site complementary to the second primer, the nucleic acid sequence complementary to the target nucleic acid sequence, and a nucleic acid sequence complementary to the first primer binding site.

20. The method of claim 19, wherein the first extension strand comprises in 5′

to 3′

order;

the first primer, the target nucleic acid sequence, and a nucleic acid sequence complementary to the second primer; and

the second extension strand comprises in 5′

to 3′

order;

the second primer, the nucleic acid sequence complementary to the target nucleic acid sequence, and a nucleic acid sequence complementary to the first primer.

21. The method of claim 20, further comprising removing from the second extension strand the nucleic acid sequence complementary to the first primer and removing from the first extension strand the nucleic acid sequence complementary to the second primer.

22. The method of claim 21, wherein the nucleic acid sequence complementary to the first primer and the nucleic acid sequence complementary to the second primer are removed by digestion with an enzyme comprising exonuclease activity.

23. The method of claim 22, wherein the enzyme comprising exonuclease activity is a DNA polymerase.

24. The method of claim 1, wherein each strand of the double stranded templated nucleic acid is generated by assembly of de novo synthesis.

25. The method of claim 1, wherein each strand of the double stranded templated nucleic acid is generated by assembly of de novo synthesis, and wherein de novo synthesis comprises synthesis of a plurality of oligonucleotides which collectively encode for a sequence of the double stranded template nucleic acid.

26. The method of claim 1, wherein the double stranded target nucleic acid has a length from about 160 to about 10,000 nucleobases.

4. (canceled)

10. (canceled)

14. (canceled)

27. A method for amplifying a template nucleic acid, the method comprising:

a) providing a template nucleic acid comprising (i) a first template strand comprising in 5′

to 3′

order;

a first primer binding site and a target nucleic acid, and (ii) a second template strand comprising in 5′

to 3′

order;

a second primer binding site and a nucleic acid sequence complementary to the target nucleic acid;

b) annealing a first primer to the first primer binding site, and annealing a second primer to the second primer binding site, wherein;

the first primer comprises 3 or 4 uracil nucleobases, each uracil nucleobase distanced from another uracil nucleobase in the first primer by about 3 to about 8 non-uracil nucleobases,

the second primer comprises 3 or 4 uracil nucleobases, each uracil nucleobase distanced from another uracil nucleobase in the second primer by about 3 to about 8 non-uracil nucleobases,

one uracil nucleobase of the 3 or 4 uracil nucleobases in the first primer is a 3′

terminal uracil nucleobase bound to a preceding nucleobase in the first primer by a first nuclease resistant bond, and

one uracil nucleobase of the 3 or 4 uracil nucleobases in the second primer is a 3′

terminal uracil nucleobase bound to a preceding nucleobase in the second primer by a second nuclease resistant bond;

c) forming an amplicon nucleic acid by;

extending the first primer and the second primer to generate a first extension strand comprising the first primer and the target nucleic acid, and extending the second primer to generate a second extension strand comprising the second primer and the nucleic acid sequence complementary to the target nucleic acid; and

d) removing the first primer and the second primer from the amplicon nucleic acid.

28-41. -41. (canceled)

42. A nucleic acid library comprising a plurality of double-stranded nucleic acids, each of the plurality of the double-stranded nucleic acids comprising:

a) a first strand comprising in 5′

to 3′

order;

a first primer and a target nucleic acid, wherein the first primer comprises a first plurality of uracil nucleobases, wherein each uracil nucleobase of the first plurality of uracil nucleobases is distanced from another uracil nucleobase in the first primer by about 3 to about 8 non-uracil nucleobases, and wherein one uracil nucleobase of the first plurality of uracil nucleobases is a 3′

terminal uracil nucleobase bound to a preceding nucleobase in the first primer by a first nuclease resistant bond; and

b) a second strand comprising in 5′

to 3′

order;

a second primer and a nucleic acid sequence complementary to the target nucleic acid, wherein the second primer comprises a second plurality of uracil nucleobases, wherein each uracil nucleobase of the second plurality of uracil nucleobases is distanced from another uracil nucleobase in the second primer by about 3 to about 8 non-uracil nucleobases, and wherein one uracil nucleobase of the second plurality of uracil nucleobases is a 3′

terminal uracil nucleobase bound to a preceding nucleobase in the second primer by a second nuclease resistant bond.

43-54. -54. (canceled)

55. A universal primer comprising:

a non-naturally occurring sequence of nucleic acids comprising 3 or more uracil nucleobases;

wherein one of the 3 or more uracil nucleobases is a 3′

terminal uracil nucleobase bound to a preceding nucleobase in the universal primer by a phosphorothioate bond; and

wherein each of the 3 or more uracil nucleobases is distanced from a subsequent or a preceding uracil nucleobase in the universal primer by about 3 to about 8 non-uracil nucleobases.

56-63. -63. (canceled)