Synthesis of novel disulfide linker based nucleotides as reversible terminators for DNA sequencing by synthesis

A method of sequencing a template polynucleotide, said method comprising:

• (i) contacting a primer hybridized to the template polynucleotide with a first nucleotide analogue comprising a first reversible terminator and extending said primer with a first polymerase by incorporating the first nucleotide analogue to form an extended primer;
• (ii) contacting the extended primer with a second nucleotide analogue capable of forming at least one hydrogen bond with a complementary nucleotide of the template polynucleotide, wherein the second nucleotide analogue is associated with a detectable moiety;
• detecting the detectable moiety;
• wherein the second nucleotide analogue is not incorporated into the extended primer;
• (iii) and contacting the extended primer with a third nucleotide analogue comprising a second reversible terminator and extending said extended primer with a polymerase by incorporating the third nucleotide analogue, wherein said first reversible terminator and said second nucleotide analogue are removed prior to step (iii);
• and
• (iv) repeating steps (i), (ii), and (iii), thereby sequencing the template polynucleotide.

A method of sequencing, said method comprising:

• contacting a primer oligonucleotide hybridized to a template polynucleotide with a first nucleotide analogue and a first polymerase thereby binding said first nucleotide analogue to a complementary nucleotide of said template polynucleotide, wherein said first nucleotide analogue is associated with a detectable moiety and comprises a moiety capable of preventing the first polymerase from catalyzing a polymerase reaction, and is not incorporated into the primer oligonucleotide;
• detecting the detectable moiety;
• removing the first nucleotide analogue;
• followed by
• contacting the primer oligonucleotide with a second nucleotide analogue and a second polymerase thereby binding said second nucleotide analogue to a complementary nucleotide of said template polynucleotide and incorporating said second nucleotide analogue into the primer oligonucleotide, wherein the second nucleotide analogue is not associated with a detectable moiety.

A method of sequencing, said method comprising:

• contacting a primer oligonucleotide hybridized to a template polynucleotide with a first nucleotide analogue and a first polymerase thereby binding said first nucleotide analogue to a complementary nucleotide of template polynucleotide, wherein said first nucleotide analogue is associated with a detectable moiety and does not form a phosphodiester bond with the primer oligonucleotide;
• detecting the detectable moiety;
• removing the first nucleotide analogue;
• followed by
• contacting the primer oligonucleotide with a second nucleotide analogue and a second polymerase thereby binding said second nucleotide analogue to a complementary nucleotide of said template polynucleotide and incorporating said second nucleotide analogue into the primer oligonucleotide, wherein the second nucleotide analogue is not associated with a detectable moiety, and wherein the first nucleotide analogue and the second nucleotide analogue comprise the same nucleobase type.