COMPOSITIONS AND METHODS FOR TERMINATING A SEQUENCING REACTION AT A SPECIFIC LOCATION IN A TARGET DNA TEMPLATE

• US 20100311055A1
• Filed: 02/17/2010
• Published: 12/09/2010
• Est. Priority Date: 01/28/2005
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: CHEN, Shiaw-Min, Brandis, John
• Application Number:
  US12/707,629
• Publication Number:
  US 20100311055A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis

Abstract

Compositions and methods for sequencing a template polynucleotide comprising a sequence of interest are provided herein. The compositions and methods employ at least one blocking probe that is designed to bind in a sequence-specific manner to a blocking sequence such that primer extension beyond the site where the blocking probe binds is reduced or prevented.

Claims

1. A method of sequencing a nucleic acid, comprising the steps of:

a) forming a first, second, third, and fourth reaction mixture such that;

(i) the first reaction mixture comprises a sequencing primer and a terminating nucleotide complementary to adenosine;

(ii) the second reaction mixture comprises a sequencing primer and a terminating nucleotide complementary to cytosine;

(iii) the third reaction mixture comprises a sequencing primer and a terminating nucleotide complementary to guanosine; and

,

(iv) the fourth reaction mixture comprises a sequencing primer and a terminating nucleotide complementary to thymidine or uridine;

b) enzymatically extending the sequencing primer in the first, second, third and fourth reaction mixtures in the presence of a composition comprising a template polynucleotide, a polymerase, a mixture of nucleoside triphosphates suitable for enzymatic primer extension, and at least one blocking probe to form a plurality of differently-sized primer extension products, wherein said template polynucleotide comprises, in a 3′

to 5′

direction, a priming sequence complementary to the sequencing primer, a sequence of interest, and a blocking sequence complementary to the blocking probe;

c) separating the primer extension products based upon their sizes; and

d) determining therefrom the sequence of the sequence of interest.

2. The method according to claim 1 in which the sequencing primer comprises a detectable label such that the primer extension products are detected based upon detection of said label.

3. The method according to claim 1 in which at least one of the nucleoside triphosphates in each of the four different reaction mixtures comprises a detectable label such that the primer extension products are detected based upon detection of said label.

4. The method according to claim 1 in which the blocking probe is selected from DNA, RNA, LNA, PNA or a chimera oligo.

5. The method according to claim 4 in which the blocking probe is PNA.

6. The method according to claim 1 in which the melting temperature (T_m) of the blocking probe is between 1°

C. to 10°

C. greater than the temperature at which the polymerase used in the sequencing reaction begins to extend the primer.

7. A method of sequencing an amplicon, comprising the steps of:

a) providing an amplicon comprising a sequence of interest;

b) amplifying said sequence of interest in the presence of a polymerase, a forward amplification primer, and a first reverse amplification primer, wherein said forward amplification comprises a 5′

tail sequence complementary to a sequencing primer and said first reverse amplification comprises a sequence complementary to the sequence of interest;

c) linearly amplifying the amplification product of step (b) in the presence of a second reverse amplification primer to form a sequencing template, wherein said second reverse amplification primer comprises a 5′

tail sequence complementary to a blocking probe, and said sequencing template comprises in a 3′

to 5′

direction, the priming sequence, the sequence of interest and the blocking sequence; and

,

d) performing a sequencing reaction according to claim 1.

8. A capillary electrophoretic method for rapidly sequencing two or more nucleic acid samples comprising the steps of:

a) independently generating a population of primer extension products within a selected size range for each nucleic acid sample to be analyzed according to claim 1;

b) injecting an aliquot of the first sample onto a capillary;

c) separating the primer extension products for the first sample;

d) detecting the separated primer extension products from the first sample;

e) injecting an aliquot of the second sample onto a capillary, wherein the capillary is not flushed prior to the injection of the second sample;

f) separating the primer extension products for the second sample; and

g) detecting the separated primer extension products from the second sample.

9. A method according to claim 8 further comprising repeating steps (e) through (g) from 1 to 8 times with subsequent nucleic acid samples.

10. A method of sequencing a nucleic acid, comprising the steps of:

a) forming a reaction mixture comprising a sequencing primer and a set of four different terminating nucleotides, each of which terminates a template-dependent primer extension reaction at a different template nucleotide and comprises a fluorescent dye capable of producing a detectable, spectrally resolvable fluorescent signal;

b) enzymatically extending the sequencing primer in the reaction mixture in the presence of a composition comprising a template polynucleotide, a polymerase, a mixture of nucleoside triphosphates suitable for enzymatic primer extension, and at least one blocking probe to form a plurality of differently-sized fluorescently labeled primer extension products, wherein said template polynucleotide comprises, in a 3′

to 5′

direction, a priming sequence complementary to the sequencing primer, a sequence of interest, and a blocking sequence complementary to the blocking probe;

c) separating the fluorescently labeled primer extension products based upon their sizes;

d) detecting the fluorescently labeled extended primers based upon their spectrally resolvable fluorescent signal; and

e) determining therefrom the sequence of the sequence of interest.

11. The method according to claim 10 in which the blocking probe is selected from DNA, RNA, LNA, PNA or a chimera oligo.

12. The method according to claim 11 in which the blocking probe is PNA.

13. The method according to claim 10 in which the melting temperature (T_m) of the blocking probe is between 1°

C. to 10°

C. greater than the temperature at which the polymerase used in the sequencing reaction begins to extend the primer.

14. A method of sequencing an amplicon, comprising the steps of:

a) providing an amplicon comprising a sequence of interest;

b) amplifying said sequence of interest in the presence of a polymerase, a forward amplification primer, and a first reverse amplification primer, wherein said forward amplification comprises a 5′

tail sequence complementary to a sequencing primer and said first reverse amplification comprises a sequence complementary to the sequence of interest;

c) linearly amplifying the amplification product of step (b) in the presence of a second reverse amplification primer to form a sequencing template, wherein said second reverse amplification primer comprises a 5′

tail sequence complementary to a blocking probe, and said sequencing template comprises in a 3′

to 5′

direction, the priming sequence, the sequence of interest and the blocking sequence; and

,

d) performing a sequencing reaction according to claim 10.

15. A capillary electrophoretic method for rapidly sequencing two or more nucleic acid samples comprising the steps of:

a) independently generating a population of primer extension products within a selected size range for each nucleic acid sample to be analyzed according to claim 10;

b) injecting an aliquot of the first sample onto a capillary;

c) separating the primer extension products for the first sample;

d) detecting the separated primer extension products from the first sample;

e) injecting an aliquot of the second sample onto a capillary, wherein the capillary is not flushed prior to the injection of the second sample;

f) separating the primer extension products for the second sample; and

g) detecting the separated primer extension products from the second sample.

16. A method according to claim 15 further comprising repeating steps (e) through (g) from 1 to 8 times with subsequent nucleic acid samples.

17. A vector comprising a blocking sequence capable of hybridizing with at least one blocking probe, and a polylinker located adjacent to the blocking sequence comprising at least one restriction endonuclease site for the insertion of a sequence of interest and a priming sequence.

18. A method for terminating a sequencing reaction, comprising the steps of:

a) forming a reaction mixture comprising a sequencing primer and at least one terminating nucleotide;

b) enzymatically extending the sequencing primer in the reaction mixture in the presence of a composition comprising a template polynucleotide, a polymerase, a mixture of nucleoside triphosphates suitable for enzymatic primer extension, wherein the sequencing primer, the terminator(s), or one or more of the nucleoside triphosphates comprises a detectable label; and

,

c) terminating the primer extension products at a pre-determined location on the polynucleotide template using at least one blocking probe, wherein said template polynucleotide comprises, in a 3′

to 5′

direction, a priming sequence complementary to the sequencing primer, a sequence of interest, and a blocking sequence complementary to the blocking probe.

19. A method according to claim 18 further comprising separating the labeled primer extension products by injecting said products onto a capillary, wherein the capillary is not flushed between sequential injections of said primer extension products.

20. A method according to claim 18 in which the terminator(s) comprises a detectable label.

21. A method according to claim 18 in which the detectable label comprises a fluorescent dye.