Method for the direct, exponential amplification and sequencing of DNA molecules and its application

Abstract

A method is described for the direct, exponential amplification and sequencing (“DEXAS”) of a DNA molecule from a complex mixture of nucleic acids, wherein truncated DNA molecules as well as DNA molecules of full length are synthesized simultaneously and exponentially between two positions on the said DNA molecule, which initially contains a DNA molecule in a thermocycling reaction, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, a thermostable pyrophosphatase (optionally), deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof.

1 . Method for the direct sequencing of a DNA molecule from a complex mixture of nucleic acids in which truncated as well as DNA molecules of full length are simultaneously synthesized between two positions on the said DNA molecule in a thermocyclic reaction which initially comprises a DNA molecule, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide, wherein the thermostable DNA polymerase has a reduced discrimination against the four ddNTPs compared to wild-type Taq polymerase.  
     
     
         2 . Method for the direct sequencing of a DNA molecule in which truncated DNA molecules as well as DNA molecules of full length are simultaneously synthesized between two positions on the said DNA molecule in a thermocycling reaction which initially comprises a DNA molecule, a first primer, a second primer, a reaction buffer, a thermostable DNA polymerase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or derivatives thereof, wherein the initial ratio of the said primers to one another is larger than 1 in the said thermocycling reaction.  
     
     
         3 . Method as claimed in claims  1  and  2 , wherein the ratio of the said primers is about 2:1.  
     
     
         4 . Method as claimed in  claims 1  to  3 , wherein the said method is carried out in a single step, in a single container, vessel or tube.  
     
     
         5 . Method as claimed in one of the  claims 1  to  4 , wherein the said primer has a length of at least 25 nucleotides.  
     
     
         6 . Method as claimed in every  claim 1  to  5 , wherein the said first primer is labelled.  
     
     
         7 . Method as claimed in one of the  claims 1  to  6 , wherein the said first primer and said second primer are labelled differently.  
     
     
         8 . Method as claimed in one of the  claims 1  to  7 , wherein the thermocycling reaction additionally contains a thermostable pyrophosphatase.  
     
     
         9 . Method as claimed in one of the  claims 1  to  8 , wherein the annealing and synthesis steps of the thermocycling reaction are carried out at a temperature of at least about 62° C.  
     
     
         10 . Method as claimed in one of the  claims 1  to  9 , wherein said DNA molecule is genomic DNA.  
     
     
         11 . Method as claimed in  claim 10 , wherein the said genomic DNA is larger than or equal to 2 kb in length.  
     
     
         12 . Method as claimed in one of the  claims 1  to  11 , wherein the source of the nucleic acid molecules to be sequenced is a source selected from body fluids such as sperm, urine, blood or blood samples, hairs, a single cell, cells or fractions thereof, tissue or fractions thereof and tissue cultures.  
     
     
         13 . Method as claimed in one of the  claims 2  to  12 , wherein the said thermostable polymerase has a reduced discrimination against the 4 ddNTPs compared to wild-type Taq DNA polymerase in the buffer or under the conditions that are used for thermocycling.  
     
     
         14 . Method as claimed in one of the  claims 1  to  13 , wherein the said thermostable polymerase is a Taq DNA polymerase with a Tabor-Richardson mutation which also has no 5′-3′ exonuclease activity or a functional derivative thereof.  
     
     
         15 . Method as claimed in one of the  claims 1  to  14 , wherein the said thermostable polymerase is Taq DNA polymerase (-exo5′-3′)(F667Y) or a functional derivative thereof.  
     
     
         16 . Use of the method as claimed in one of the  claims 1  to  15  for the determination of a sequence of a nucleic acid.  
     
     
         17 . Use of the method as claimed in one of the  claims 1  to  15  for the direct sequencing of eukaryotic genomic DNA.  
     
     
         18 . Use of the method as claimed in one of the  claims 1  to  15  for the direct sequencing of human chromosomal or mitochondrial DNA.  
     
     
         19 . Use of the method as claimed in one of the  claims 1  to  15  for the direct sequencing of human RNA.  
     
     
         20 . Use of the method as claimed in one of the  claims 1  to  15  for the direct sequencing of unpurified plasmid DNA from bacterial colonies.  
     
     
         21 . Use of the method as claimed in one of the  claims 1  to  15  for the direct sequencing of unpurified single-stranded or double-stranded DNA from bacteriophages.  
     
     
         22 . Kit for the direct sequencing of a nucleic acid molecule from a complex mixture of nucleic acids containing a reaction buffer, deoxynucleotides or derivatives thereof and a dideoxynucleotide or another terminating nucleotide and a thermostable polymerase which has a reduced discrimination against ddNTPs compared to wild-type Taq DNA polymerase.  
     
     
         23 . Kit for the direct sequencing of a nucleic acid molecule containing a reaction buffer, deoxynucleotides or derivatives thereof, dideoxy-nucleotides or another terminating nucleotide, a thermostable polymerase and two primers whose ratio is larger than 1.  
     
     
         24 . Kit for sequencing a nucleic acid molecule as claimed in  claim 23 , wherein the thermostable polymerase has a reduced discrimination against ddNTPs compared to wild-type Taq DNA polymerase.