Reversible di-nucleotide terminator sequencing

• US 8,715,938 B2
• Filed: 08/05/2011
• Issued: 05/06/2014
• Est. Priority Date: 11/20/2007
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Zon, Gerald
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US13/198,901
• Publication Number:
  US 20130065225A1
• Time in Patent Office: 1,005 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides

Abstract

The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, reversible di-nucleotide compounds are employed along with cleaving reactions that remove a label and a blocking moiety. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of reversible di-nucleotide compounds. In some embodiments, the di-nucleotides do not contain conventional nucleotide triphosphates, but rather employ amino acid phosphoramidate nucleotides (AAPNs).

Claims

1. A method for determining a sequence of a template polynucleotide, the method comprising:

a) hybridizing a first primer to the template polynucleotide;

b) contacting at least one labeled reversible di-nucleotide terminator and a DNA polymerase with the first primer to extend the first primer along the template by polymerase-mediated polymerization of the labeled reversible di-nucleotide terminator to form a first extension product, wherein the labeled reversible di-nucleotide terminator includes a first nucleotide connected to a second nucleotide and includes a detectable moiety linked to the first nucleotide and includes a blocking moiety linked to the second nucleotide;

c) detecting the detectable moiety of the polymerized labeled reversible di-nucleotide terminator of step (b);

d) identifying a probe family to which the polymerized labeled reversible di-nucleotide terminator in step (b) belongs, the probe family having a plurality of labeled reversible di-nucleotide terminators of varying sequence each having the same type of a detectable moiety;

e) removing the blocking group on the polymerized labeled reversible di-nucleotide terminator of step (b) to generate a free 3′

OH group;

f) repeating steps (b), (c), (d), and optionally (e), to generate a first accumulated extension product;

g) generating a set of candidate ordered series of the probe families which correlate with an ordered series of the polymerized labeled reversible di-nucleotide terminators in steps (b), (c) and (d); and

h) determining the sequence of the template polynucleotide based at least in part on the first set of candidate ordered series of the probe families.

2. The method of claim 1, further comprising the step of removing the detectable moiety after step (c).

3. The method of claim 1, wherein the first type of nucleotide is adenosine, guanosine, cytidine, or thymidine.

4. The method of claim 1, wherein the second type of nucleotide is adenosine, guanosine, cytidine, or thymidine.

5. The method of claim 1, wherein the blocking moiety is linked to the 3C position on the second nucleotide in step (b).

6. The method of claim 1, wherein the detectable moiety is linked to the base or the 3C of the first nucleotide in step (b).

7. The method of claim 1, wherein the detectable moiety is a fluorophore.

8. The method of claim 1, wherein the polynucleotide template is immobilized to a bead.

9. The method of claim 1, further comprising polymerizing a dideoxynucleotide on the first extension product of step (b) so as to block further extension of the first or second extension product.

10. The method of claim 1, wherein the labeled reversible di-nucleotide terminator comprises an amino acid phosphoramidate nucleotide (AAPN).

11. A method for determining a sequence of a template polynucleotide, the method comprising:

a) hybridizing a first primer to the template polynucleotide;

b) contacting at least one labeled reversible di-nucleotide terminator and a DNA polymerase with the first primer to extend the first primer along the template by polymerase-mediated polymerization of the labeled reversible di-nucleotide terminator to form a first extension product, wherein the labeled reversible di-nucleotide terminator includes a first nucleotide connected to a second nucleotide and includes a detectable moiety linked to the first nucleotide and includes a blocking moiety linked to the second nucleotide, and wherein the labeled reversible di-nucleotide terminator is a member of a probe family having a plurality of labeled reversible di-nucleotide terminators each having the same type of a first nucleotide linked to the same type of a detectable moiety and having a different type of a second nucleotide;

c) detecting the detectable moiety of the polymerized labeled reversible di-nucleotide terminator of step (b);

d) identifying the probe family to which the polymerized labeled reversible di-nucleotide terminator in step (b) belongs;

e) removing the blocking group on the polymerized labeled reversible di-nucleotide terminator of step (b) to generate a free 3′

OH group;

f) repeating steps (b), (c), (d), and optionally (e), to generate a first accumulated extension product;

g) generating a first set of candidate ordered series of the probe families which correlate with an ordered series of the polymerized labeled reversible di-nucleotide terminators in steps (b), (c) and (d); and

h) determining the sequence of the template polynucleotide based at least in part on the first set of candidate ordered series of the probe families.

12. The method of claim 11, further comprising the step of removing the detectable moiety after step (c).

13. The method of claim 11, further comprising polymerizing a dideoxynucleotide on the first extension product of step (b) so as to block further extension of the first or second extension product.

14. A method for determining a sequence of a template polynucleotide, the method comprising:

a) hybridizing a first primer to the template polynucleotide;

b) contacting at least one labeled reversible di-nucleotide terminator and a DNA polymerase with the first primer to extend the first primer along the template by polymerase-mediated polymerization of the labeled reversible di-nucleotide terminator to form a first extension product, wherein the labeled reversible di-nucleotide terminator includes a first nucleotide connected to a second nucleotide and includes a detectable moiety linked to the first nucleotide and includes a blocking moiety linked to the second nucleotide;

c) detecting the detectable moiety of the polymerized labeled reversible di-nucleotide terminator of step (b);

d) identifying a probe family to which the polymerized labeled reversible di-nucleotide terminator in step (b) belongs, the probe family having a plurality of labeled reversible di-nucleotide terminators of varying sequence each having the same type of a detectable moiety;

e) removing the blocking group on the polymerized labeled reversible di-nucleotide terminator of step (b) to generate a free 3′

OH group;

f) repeating steps (b), (c), (d), and optionally (e), to generate a first accumulated extension product;

g) generating a set of candidate ordered series of the probe families which correlate with an ordered series of the polymerized labeled reversible di-nucleotide terminators in steps (b), (c) and (d); and

h) decoding the first set of candidate ordered series of the probe families to identify the most likely member of the first set of candidate ordered series.

15. The method of claim 14, further comprising the step of removing the detectable moiety after step (c).

16. The method of claim 14, further comprising polymerizing a dideoxynucleotide on the first extension product of step (b) so as to block further extension of the first or second extension product.