Methods, compositions, and kits for amplifying and sequencing polynucleotides

Abstract

In one aspect, there are provided methods of amplifying and sequencing a polynucleotide. In some embodiments, the method includes (a) amplifying the polynucleotide with at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and one or more terminating nucleotides, the incubation being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase; (b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase; (c) evaluating the extended sequencing primers in order to determine the sequence of the polynucleotide. The reactions at the first and second temperatures can be carried out in a single reaction vessel. In other aspects, compositions and kits for carrying out the methods are also provided.

1 . A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of: 
 a) incubating the polynucleotide with a composition comprising: at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, and four different terminating nucleotides, each of which terminates template-dependent primer extension at a different template nucleotide, the incubating being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase;    b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase;    c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.    
     
     
         2 . The method of  claim 1  wherein each different terminating nucleotide is labeled with a different, distinguishable label.  
     
     
         3 . The method of  claim 2  wherein said determining comprises separating said extended sequencing primers based upon their sizes; detecting the label on said extended sequencing primers; and determining therefrom a sequence of the polynucleotide.  
     
     
         4 . The method of  claim 1  wherein said sequencing primer is labeled.  
     
     
         5 . The method of  claim 1  wherein said at least one amplification primer is resistant to at least one of 3′ exonuclease activity and 5′ exonuclease activity.  
     
     
         6 . The method of  claim 1  wherein said at least one amplification primer is a sequence specific primer.  
     
     
         7 . The method of  claim 1  wherein said at least one amplification primer is a random primer.  
     
     
         8 . The method of  claim 7  wherein said at least one amplification primer comprises a plurality of sequence specific primers.  
     
     
         9 . The method of  claim 8  wherein said at least one amplification primer comprises a plurality of random primers.  
     
     
         10 . The method of  claim 9  wherein each of said primers comprising the plurality are of equal length and wherein the length is in the range of from 4 to 25 bases.  
     
     
         11 . The method of  claim 10  wherein each of said primers is a hexamer.  
     
     
         12 . The method of  claim 9  wherein the plurality of random primers comprises the complete set of hexamers composed of bases A, G, C and T.  
     
     
         13 . The method of  claim 12  in which the plurality of primers comprises a subset of the complete set of hexamers composed of A, G, C and T.  
     
     
         14 . The method of  claim 1  wherein said at least one amplification primer is a polynucleotide, a polynucleotide analog or a chimera thereof.  
     
     
         15 . The method of  claim 1  wherein said sequencing primer comprises a modified nucleoside base.  
     
     
         16 . The method of  claim 1  wherein said at least one amplification primer comprises a modified nucleoside base.  
     
     
         17 . The method of  claim 1  wherein said at least one amplification primer and/or said sequencing primer is DNA, RNA, LNA, PNA or a chimera thereof.  
     
     
         18 . The method of  claim 1  wherein said sequencing primer is in the range of 10-50 bases in length.  
     
     
         19 . The method of  claim 1  wherein said sequencing primer is a polynucleotide, a polynucleotide analog or a chimera thereof.  
     
     
         20 . The method of  claim 2  wherein each of the four different terminating nucleotides is labeled with a fluorescent label.  
     
     
         21 . The method of  claim 1  wherein the polynucleotide is the product of an amplification reaction.  
     
     
         22 . The method of  claim 1  wherein the sequences and lengths of said at least one amplification and sequencing primer are such that the annealing temperature between said polynucleotide and said at least one amplification primer is at least about 5° C. lower than the annealing temperature of a duplex formed between said sequencing primer and the amplification product of step (a).  
     
     
         23 . The method of  claim 1  wherein the sequences and lengths of said at least one amplification and sequencing primer are such that the annealing temperature between said polynucleotide and said at least one amplification primer is at least about 10° C. lower than the annealing temperature of a duplex formed between said sequencing primer and the amplification product of step (a).  
     
     
         24 . The method of  claim 1  wherein the sequences and lengths of said at least one amplification and said sequencing primer are such that said T m  of a duplex formed between said polynucleotide and said at least one amplification primer is at least about 5° C. lower than said T m  of a duplex formed between said sequencing primer and the amplification product of step (a).  
     
     
         25 . The method of  claim 1  in which the amplifying is carried out in the presence of four different terminating nucleoside triphosphates, each of which terminates primer extension at a different one of A, C, G/I, or T/U and which is labeled with a different, distinguishable label.  
     
     
         26 . A method of amplifying and sequencing at least a region of a polynucleotide, said method comprising the steps of: 
 a) incubating the polynucleotide with a composition comprising: at least one amplification primer, a processive amplification polymerase, a sequencing primer, a sequencing polymerase, deoxynucleoside triphosphates suitable for template-dependent primer extension, a terminating nucleotide, the incubating being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase, wherein said sequencing primer is labeled with a detectable label;    b) incubating the product of step (a) at a second temperature suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase, wherein step (a) and step (b) are repeated in four separate incubations in the presence of a terminating nucleotide suitable for terminating primer extension at A, C, G/I, or T/U, respectively;    c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.    
     
     
         27 . A method of amplifying and sequencing a polynucleotide, said method comprising the steps of: 
 a) amplifying at least a region of a polynucleotide with a processive polymerase and at least one amplification primer at a first temperature in the presence of a sequencing primer, a sequencing polymerase, nucleoside triphosphates suitable for template-dependent primer extension, four different terminating nucleotides, the amplifying being carried out at a first temperature suitable for amplifying the polynucleotide with the processive amplification polymerase;    b) forming at a second temperature a plurality of differently-sized extended and labeled sequencing primers, wherein said second temperature is suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase;    c) determining a sequence of said polynucleotide by evaluating said plurality of differently-sized extended sequencing primers.