Alternative nucleotide flows in sequencing-by-synthesis methods

• US 10,392,660 B2
• Filed: 01/12/2017
• Issued: 08/27/2019
• Est. Priority Date: 06/11/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Schultz, Jonathan, Davidson, John
• Primary Examiner(s): Hammell, Neil P
• Application Number:
  US15/404,735
• Publication Number:
  US 20170121766A1
• Time in Patent Office: 957 Days
• Field of Search: None
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

A method of sequencing a polynucleotide strand can include providing the polynucleotide strand with a primer annealed thereto and a polymerase operably bound to the polynucleotide strand; successively exposing the polynucleotide strand to the flow of four different dNTPs according to a first predetermined ordering; and successively exposing the polynucleotide strand to the flow of four different dNTPs according to a second predetermined ordering, wherein the second predetermined ordering is different from the first predetermined ordering.

Claims

1. A method for sequencing a template polynucleotide strand comprising:

(a) disposing a plurality of template polynucleotide strands into a plurality of reaction chambers, such that each reaction chamber has a template polynucleotide strand of the plurality having a sequencing primer hybridized thereto and a polymerase operably bound thereto;

(b) introducing a known nucleoside triphosphate into each reaction chamber according to a predetermined ordering of dNTP reagent flows;

(c) detecting sequential incorporation at the 3′

end of the sequencing primer of one or more nucleoside triphosphates if the known nucleoside triphosphate is complementary to corresponding nucleotides in the template polynucleotide strands;

(d) washing away unincorporated nucleoside triphosphates from the reaction chamber; and

(e) repeating steps (b) through (d) until a desired template length of the plurality of template polynucleotide strands is sequenced;

wherein the predetermined ordering of dNTP reagent flows comprises an alternate ordering which is not a continuous and ordered repeat of four different dNTP reagent flows, the alternate ordering comprising a same number of flows for each of four different dNTP reagent flows, and at least one flow of each kind of dNTP reagent followed by a flow of the same kind of dNTP reagent after a single intervening flow of a different kind of dNTP reagent, and wherein repetition of the alternate ordering successively occurs at least once to sequence the desired template length.

2. The method of claim 1, wherein repetition of the alternate ordering successively occurs at least twice.

3. The method of claim 1, wherein the predetermined ordering of dNTP reagent flows comprises one of 20, 24, 28 and 32 dNTP reagent flows.

4. A method of sequencing a polynucleotide strand, comprising:

providing the polynucleotide strand with a primer annealed thereto and a polymerase operably bound to the polynucleotide strand;

successively exposing the polynucleotide strand to a first series of flows of four different dNTPs according to a first predetermined ordering; and

successively exposing the polynucleotide strand to a second series of flows of four different dNTPs according to a second predetermined ordering, wherein;

the second predetermined ordering is different from the first predetermined ordering,

a same number of flows of each of the four different dNTPs occurs in the first series of flows and the second series of flows, and

repetition of the first and second predetermined orderings successively occurs at least once to sequence a desired template length,

wherein one of the first or second predetermined orderings comprises a flow of a nucleotide N, which is followed directly by a flow of a different nucleotide W, which is followed directly by a flow of the same nucleotide N, wherein the N-W-N flow is further followed directly by a flow of a nucleotide Z that is different from N and W.

5. The method of claim 4, wherein the second predetermined ordering comprises an alternate ordering which is not a continuous repeat of the first predetermined ordering of the four different dNTPs.

6. The method of claim 4, further comprising successively exposing the polynucleotide strand to a third series of flows of four different dNTPs according to a third predetermined ordering, wherein the third predetermined ordering comprises an alternate ordering which is not a continuous repeat of the first predetermined ordering of the four different dNTPs or of the second predetermined ordering of the four different dNTPs.

7. The method of claim 4, further comprising successively exposing the polynucleotide strand to a third series of flows of four different dNTPs according to the first predetermined ordering after successively exposing the polynucleotide strand to the second series of flows of four different dNTPs according to the second predetermined ordering.

8. The method of claim 4, wherein repetition of each of the first and second predetermined orderings successively occurs at least twice.

9. The method of claim 4, wherein each of the first and second predetermined orderings of dNTP reagent flows comprises one of 20, 24, 28 and 32 dNTP reagent flows.