Compositions and methods for nucleic acid amplification

Patent metadata

Field	Value
Publication number	US-2016333340-A1
Application number	US-201615151316-A
Country	US
Kind code	A1
Filing date	May 10, 2016
Priority date	May 11, 2015
Publication date	Nov 17, 2016
Grant date	—

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.

First claim

What is claimed is:

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.

4 . (canceled)

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, εA (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.

10 . (canceled)

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.

14 . (canceled)

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 co

Assignees

• Twist Bioscience Corp

Inventors

• Wu Cheng-Hsien

Classifications

• C12Q1/6853 Primary

  using modified primers or templates · CPC title

• C12N15/1093 Primary

  General methods of preparing gene libraries, not provided for in other subgroups · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C07H21/04 Primary

  with deoxyribosyl as saccharide radical · CPC title