Compositions and methods for terminating a sequencing reaction at a specific location in a target dna template

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.

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.