Reversible di-nucleotide terminator sequencing
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).
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, 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);
h) removing the first accumulated extension product;
i) hybridizing a second primer to a position on the template polynucleotide that is shifted relative to the position of the hybridized first primer;
j) extending the second primer along the template by polymerase-mediated polymerization of a labeled reversible di-nucleotide terminator to form a second 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;
k) detecting the detectable moiety of the polymerized labeled reversibly di-nucleotide terminator of step (j);
l) identifying the probe family to which the polymerized labeled reversible di-nucleotide terminator in step (j) belongs;
m) removing the blocking group on the polymerized labeled reversible di-nucleotide terminator of step (j) to generate a free 3′ OH group;
n) repeating steps (j), (k), (l), and optionally (m), to generate a second accumulated extension product;
o) generating a second 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 (j), (k) and (l);
p) decoding the first and second sets of candidate ordered series of the probe families to identify the most likely member of the first or second sets of candidate ordered series to determine the sequence of the template polynucleotide.
2. The method of claim 1 further comprising the step of removing the detectable moiety after step (c) or (k).
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) or (j).
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) or (j).
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 , wherein the polynucleotide template is prepared by:
a) amplifying the template polynucleotide in a compartment of an emulsion in the presence of an amplification primer immobilized to a microparticle so that a microparticle having a clonal population of template attached thereto is produced;
b) recovering the microparticle from the emulsion; and,
c) embedding the microparticle in or on a semi-solid support.
10. The method of claim 1 further comprising polymerizing a dideoxynucleotide on the first extension product of step (b) or the second extension product of step (j) so as to block further extension of the first or second extension product.
11. The method of claim 1 , wherein the labeled reversible di-nucleotide terminator comprises an amino acid phosphoramidate nucleotide (AAPN).