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 
 at least one terminating nucleotide, wherein a first annealing temperature of a duplex formed between the polynucleotide and the at least one amplification primer is lower than a second annealing temperature of a duplex formed between the sequencing primer and an amplification product; 
   b) maintaining a first incubation temperature isothermally at a temperature suitable for amplifying the polynucleotide with the amplification polymerase;   c) elevating the incubation temperature to one or more temperatures suitable for forming a plurality of differently-sized extended sequencing primers with the sequencing polymerase, wherein the one or more temperatures are above the first annealing temperature; and   d) determining a sequence of the polynucleotide by evaluating the plurality of differently-sized extended sequencing primers.   
     
     
         2 . The method of  claim 1  wherein the at least one terminating nucleotide is labeled with a distinguishable label. 
     
     
         3 . The method of  claim 2  wherein the determining comprises separating said extended sequencing primers based upon their sizes; detecting the label on the extended sequencing primers; and determining therefrom a sequence of the polynucleotide. 
     
     
         4 . The method of  claim 1  wherein the at least one amplification primer is resistant to at least one of 3′ exonuclease activity and 5′ exonuclease activity. 
     
     
         5 . The method of  claim 1  wherein the at least one amplification primer is a sequence specific primer. 
     
     
         6 . The method of  claim 1  wherein the at least one amplification primer is a random primer. 
     
     
         7 . The method of  claim 6  wherein the at least one amplification primer comprises a plurality of sequence specific primers. 
     
     
         8 . The method of  claim 7  wherein the at least one amplification primer comprises a plurality of random primers. 
     
     
         9 . The method of  claim 8  wherein each of the primers comprising the plurality are of equal length and wherein the length is in the range of from 4 to 25 bases. 
     
     
         10 . The method of  claim 8  wherein each of the primers is a hexamer. 
     
     
         11 . The method of  claim 8  wherein the plurality of random primers comprises the complete set of hexamers composed of bases A, G, C and T. 
     
     
         12 . The method of  claim 11  in which the plurality of primers comprises a subset of the complete set of hexamers composed of A, G, C and T. 
     
     
         13 . The method of  claim 1  wherein the at least one amplification primer is a polynucleotide, a polynucleotide analog or a chimera thereof. 
     
     
         14 . The method of  claim 1  wherein the at least one amplification primer comprises a modified nucleoside base. 
     
     
         15 . The method of  claim 1  wherein the at least one amplification primer and/or said sequencing primer is DNA, RNA, LNA, PNA or a chimera thereof. 
     
     
         16 . The method of  claim 1  wherein the sequencing primer is in the range of 10-50 bases in length. 
     
     
         17 . The method of  claim 1  wherein the sequencing primer is a polynucleotide, a polynucleotide analog or a chimera thereof. 
     
     
         18 . The method of  claim 2  wherein the at least one terminating nucleotide is labeled with a fluorescent label. 
     
     
         19 . The method of  claim 1  wherein the polynucleotide is the product of an amplification reaction. 
     
     
         20 . 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. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein the amplification polymerase and the sequencing polymerase are the same polymerase. 
     
     
         23 . The method of  claim 1 , wherein the processive-amplification polymerase has strand displacement activity. 
     
     
         24 . The method of  claim 1 , wherein the processive amplification polymerase is φ29 polymerase or Bst DNA polymerase. 
     
     
         25 . The method of  claim 1 , wherein the processive amplification polymerase is an exo(−)Bst DNA polymerase. 
     
     
         26 . A kit comprising:
 a processive polymerase;   a sequencing polymerase;   at least one amplification primer;   a sequencing primer;   deoxynucleoside triphosphates suitable for template-dependent primer extension; and   at least one terminating nucleotide which is labeled with a distinguishable label, wherein the at least one amplification primer is capable of annealing to at least a region of the target polynucleotide at a first temperature; and the sequencing primer is capable of annealing to the amplification product obtained from the amplification of the target polynucleotide at a second temperature which is not suitable for annealing of the at least one amplification primer to said amplification product.