Nucleic acid amplification with direct sequencing

Abstract

This invention provides methods of amplifying a sequence of interest present within a nucleic acid molecule. In addition, this invention provides a method of determining the nucleotide sequence of a sequence of interest present within a nucleic acid molecule (e.g. GAWTS and RAWTS) which can be used to sequence tissue specific genes (e.g. tsRAWTS) and genes accross species (e.g. zooRAWTS). In addition, this invention provides a method of synthesizing a polypeptide encoded for by a nucleic acid molecule (RAWIT). Further, the subject invention provides a method of determining an internal nucleotide sequence present within a nucleic acid molecule, and a method of determining a terminal nucleotide sequence present within a nucleic acid molecule (e.g. PLATS). Also provided for is a method of determining the nucleotide sequence of sequences present within a nucleic acid molecule which are adjacent to areas of known sequence (e.g ASWATS) and a method of determining the nucleotide sequence of sequences present within a nucleic acid molecule and a method of detecting point mutation or polymorphism (e.g. PASA) which can be used in low cost methods of carrier testing and prenatal diagnosis. Lastly, this invention provides methods for determining the exonic nucleotide sequence of a gene as well as methods of detecting genomic mutations.

What is claimed is:  
     
         1 . A method of amplifying a sequence of interest present within a nucleic acid molecule which comprises: 
 A) obtaining a sample of the nucleic acid molecule which contains the sequence of interest;    B) if the nucleic acid molecule is a single-stranded RNA molecule, treating the sample from step (A) so as to prepare a sample containing a DNA molecule which contains a sequence complementary to the sequence of interest;    C) treating the sample from step (A) if the nucleic acid molecule is a DNA molecule or the sample from step (B) if the nucleic acid molecule is a single-stranded RNA molecule so as to obtain a further sample containing a single-stranded DNA molecule which contains a sequence complementary to the sequence of interest;    D) contacting the further sample from step (C) under hybridizing conditions with one oligonucleotide primer which includes at least (a) a promoter and (b) a nucleic acid sequence present within the nucleic acid molecule which contains the sequence of interest, which primer sequence is located adjacent to, and 5′ of, the sequence of interest, so that the oligonucleotide primer hybridizes with the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest;    E) treating the resulting sample containing the single-stranded DNA molecule to which the oligonucleotide primer is hybridized from step (D) with a polymerase under polymerizing conditions so that a DNA extension product of the oligonucleotide primer is synthesized, which DNA extension product contains the sequence of interest;    F) treating the sample from step (E) so as to separate the DNA extension product from the single-stranded DNA molecule on which it was synthesized and thereby obtain single-stranded DNA molecules;    G) contacting the resulting sample from step (F) containing the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest under hybridizing conditions, with one oligonucleotide primer, which includes at least (a) a promoter and (b) a nucleic acid sequence located adjacent to, and 5′ of, the sequence of interest, so that the oligonucleotide primer hybridizes with the single-stranded DNA molecule present in the sample which contains the sequence complementary to the sequence of interest;    H) treating the sample containing the single-stranded DNA molecule to which the oligonucleotide primer is hybridized from step (G) with a polymerase so as to synthesize a further DNA extension product containing the sequence complementary to the sequence of interest;    I) repeating steps (F) through (H), as desired;    J) contacting the sample from step (I) with an RNA polymerase which initiates polymerization from the promoter present, under polymerizing conditions, so as to obtain multiple RNA transcripts of each DNA extension product which contains the sequence complementary to the sequence of interest, thereby amplifying the sequence of interest.    
     
     
         2 . A method of amplifying a sequence of interest present within a nucleic acid molecule which comprises: 
 A) obtaining a sample of the nucleic acid molecule which contains the sequence of interest;    B) if the nucleic acid molecule is a single-stranded RNA molecule, treating the sample from step (A) so as to prepare a sample containing a DNA molecule which contains a sequence complementary to the sequence of interest;    C) treating the sample from step (A) if the nucleic acid molecule is a DNA molecule or the sample from step (B) if the nucleic acid molecule is a single-stranded RNA molecule so as to obtain a further sample containing a single-stranded DNA molecule which contains a sequence complementary to the sequence of interest;    D) contacting the further sample from step (C) under hybridizing conditions with two or more oligonucleotide primers at least one of which includes at least (a) a promoter and (b) a nucleic acid sequence present within the nucleic acid molecule which contains the sequence of interest, which primer sequence is located adjacent to, and 5′ of, the sequence of interest, and at least one other of which includes a nucleic acid sequence complementary to a sequence present within the nucleic acid molecule which contains the sequence of interest, which primer, sequence is located adjacent to, and 5′ of, the nucleic acid sequence complementary to the sequence within the nucleic acid molecule which contains the sequence of interest, so that at least one of the oligonucleotide primers hybridizes with the single-stranded DNA molecule present in the sample which contains the sequence complementary to the sequence of interest, and at least one other of the oligonucleotide primers hybridizes with the single-stranded DNA molecule which contains the sequence of interest;    E) treating the resulting sample containing the single-stranded DNA molecules to which the oligonucleotide primers are hybridized from step (D) with a polymerase under polymerizing conditions so that DNA extension products of the oligonucleotide primers are synthesized, some of which DNA extension products contain the sequence of interest and some of which DNA extension products contain the sequence complementary to the sequence of interest;    F) treating the sample from step (E) so as to separate the DNA extension products from the single-stranded DNA molecules on which they were synthesized and thereby obtain single-stranded DNA molecules;    G) contacting the resulting sample from step (F) containing the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest under hybridizing conditions, with two or more oligonucleotide primers at least one which includes at least (a) a promoter and (b) a nucleic acid sequence located adjacent to, and 5′ of, the sequence of interest, and at least one other of which includes a nucleic acid sequence complementary to a sequence present within the nucleic acid molecule which contains the sequence of interest, which primer sequence is located adjacent to, and 5′ of, the nucleic acid sequence complementary to the sequence within the nucleic acid molecule which contains the sequence of interest, so that at least one of the oligonucleotide primers DNA molecule present in the sample which contains the sequence complementary to the sequence of interest, and at least one other of the oligonucleotide primers hybridizes with the single-stranded DNA molecule which contains the sequence of interest;    H) at least treating the sample containing the single-stranded DNA molecules to which the oligonucleotide primers are hybridized from step (G) with polymerase so as to synthesize further DNA extension products, some of which DNA extension products contain the sequence of interest and some of which DNA extension products contain the sequence complementary to the sequence of interest;    I) repeating steps (F) through (H), as desired;    J) contacting the sample from step (I) with an RNA polymerase which initiates polymerization from the promoter present, under polymerizing conditions, so as to obtain multiple RNA transcripts of each DNA extension product which contains the sequence complementary to the sequence of interest, thereby amplifying the sequence of interest.    
     
     
         3 . A method of  claim 1  or  2 , wherein the nucleic acid molecule containing the sequence of interest comprises double-stranded DNA.  
     
     
         4 . A method of  claim 3 , wherein the double-stranded DNA comprises genomic DNA.  
     
     
         5 . A method of  claim 1  or  2 , wherein the nucleic acid molecule containing the sequence of interest comprises cDNA.  
     
     
         6 . A method of  claim 1  or  2 , wherein the nucleic acid molecule containing the sequence of interest comprises RNA.  
     
     
         7 . A method of  claim 6 , wherein the nucleic acid molecule containing the sequence of interest comprises mRNA.  
     
     
         8 . A method of  claim 1  or  2 , wherein the sample comprises a biological sample.  
     
     
         9 . A method of  claim 8 , wherein the biological sample is a cell sample.  
     
     
         10 . A method of  claim 8 , wherein the biological sample is a tissue sample.  
     
     
         11 . A method of  claim 10 , wherein the tissue sample is blood.  
     
     
         12 . A method of  claim 1  or  2 , wherein the promoter is a phage promoter.  
     
     
         13 . A method of  claim 12 , wherein the phage promoter is a T7 promoter.  
     
     
         14 . A method of  claim 12 , wherein the phage promoter is a T3 promoter.  
     
     
         15 . A method of  claim 12 , wherein the phage promoter is an SP6 promoter.  
     
     
         16 . A method of  claim 1  or  2 , wherein in step (D) the oligonucleotide primer which hybridizes with the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest comprises a T7 promoter and in step (J) the RNA polymerase comprises a T7 RNA polymerase.  
     
     
         17 . A method of  claim 1  or  2 , wherein in step (D) the oligonucleotide primer which hybridizes with the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest comprises a T3 promoter and in step (J) the RNA polymerase comprises a T3 RNA polymerase.  
     
     
         18 . A method of  claim 1  or  2 , wherein in step (D) the oligonucleotide primer which hybridizes with the single-stranded DNA molecule which contains the sequence complementary to the sequence of interest comprises a SP6 promoter and in step (J) the RNA polymerase comprises a SP6 RNA polymerase.  
     
     
         19 . A method of determining the nucleotide sequence of a sequence of interest present within a nucleic acid molecule which comprises: 
 a) amplifying the sequence of the nucleic acid molecule to be determined using the method of  claim 1  or  2 ;    b) treating the sample from step (J) of  claim 1  or  2 , under conditions such that a primer hybridizes to the RNA transcript;    c) contacting the sample from step (b) with a polymerase under polymerizing conditions such that a single-stranded nucleic acid molecule which is complementary to the RNA transcript is synthesized; and    d) determining the nucleotide sequence of the single-stranded nucleic acid molecule obtained in step (c) thereby determining the nucleotide sequence of a sequence of interest.    
     
     
         20 . A method of  claim 19 , wherein the polymerase is reverse transcriptase.  
     
     
         21 . A method of  claim 19 , wherein in step (d) the determining comprises enzymatic sequencing.  
     
     
         22 . A method of  claim 21 , wherein the enzymatic sequencing comprises Sanger dideoxy sequencing.  
     
     
         23 . A method of  claim 19 , wherein in step (d) the determining comprises chemical sequencing.  
     
     
         24 . A method of  claim 23 , wherein the chemical sequencing comprises Maxam Gilbert sequencing.  
     
     
         25 . A method of  claim 19 , wherein in step (d) the determining comprises both chemical and enzymatic sequencing.  
     
     
         26 . A method of  claim 25 , wherein the sequencing comprises the use of phosphorothioate.  
     
     
         27 . A method of determining the nucleotide sequence of a sequence of interest present within a nucleic acid molecule which comprises: 
 a) amplifying the amount of the sequence of interest present within a nucleic acid molecule;    b) if the sequence generated in step (a) is double-stranded, treating the molecule to generate single-stranded nucleic acid molecules;    c) determining the sequence of the single-stranded nucleic acid molecules of either step (a) or (b) thereby determining the nucleotide sequence of the sequence of interest.    
     
     
         28 . A method of  claim 27 , wherein in step (c) the determining comprises enzymatic sequencing.  
     
     
         29 . A method of  claim 28 , wherein the enzymatic sequencing comprises Sanger dideoxy sequencing.  
     
     
         30 . A method of  claim 27 , wherein in step (c) the determining comprises chemical sequencing.  
     
     
         31 . A method of  claim 30 , wherein the chemical sequencing comprises Maxam Gilbert sequencing.  
     
     
         32 . A method of  claim 27 , wherein in step (c) the determining comprises both chemical and enzymatic sequencing.  
     
     
         33 . A method of  claim 32 , wherein the sequencing comprises the use of phosphorothioate.  
     
     
         34 . A method of synthesizing a polypeptide encoded for by a nucleic acid molecule which comprises: 
 a) amplifying a sequence of interest present within a nucleic acid molecule which encodes for the polypeptide to be synthesized using the method of  claim 1  or  2  wherein at least one of the oligonucleotides contains a translation initiation signal 3′ to the promoter; and    b) translating the RNA of step (a) to produce the polypeptide or fragment thereof encoded for by the nucleic acid molecule.    
     
     
         35 . A method of producing a therapeutic agent containing one or more polypeptides or fragments thereof which comprises synthesizing the polypeptide or fragment thereof by the method of  claim 34 .  
     
     
         36 . A method of determining an internal nucleotide sequence present within a nucleic acid molecule which contains promoters at both ends of the nucleic acid molecule which comprises: 
 a) cleaving the nucleic acid molecule under such conditions so as to generate fragments of the nucleic acid molecule;    b) if the fragments of the nucleic acid. molecule do not have blunt ends, treating the fragments of the nucleic acid molecule so as to generate blunt ends;    c) ligating a promoter to the blunt end of a fragment of the nucleic acid molecule obtained in step (a) or (b);    d) amplifying a sequence of the fragment of the nucleic acid molecule containing the promoter obtained in step (c);    e) transcribing the amplified fragment of the nucleic acid molecule obtained in step (d); and    f) sequencing the transcript obtained in step (e) thereby determining an internal nucleotide sequence present within nucleic acid molecule.    
     
     
         37 . A method of  claim 36 , wherein in step (c) the promoter comprises a double-stranded promoter.  
     
     
         38 . A method of  claim 36 , wherein in step (a) the cleaving comprises shearing the nucleic acid molecule.  
     
     
         39 . A method of  claim 36 , wherein in step (a) the cleaving comprises the use of a restriction endonuclease.  
     
     
         40 . A method of  claim 36 , wherein the promoters comprise phage promoters.  
     
     
         41 . A method of  claim 40 , wherein the promoters are a T7 promoter, a T3 promoter, and a SP6 promoter.  
     
     
         42 . A method of determining a terminal nucleotide sequence present within a nucleic acid molecule which comprises: 
 a) digesting a nucleic acid molecule with one or more restriction enzymes to generate fragments of the nucleic acid molecule having either blunt ends, or 5′ overhangs;    b) if the nucleic acid fragment has a 5′ overhangs, treating the fragment of the nucleic acid molecule obtained in step (a) to generate blunt ends;    c) contacting the fragment obtained in step (b) with two different primer sequences containing different promoters under hybridizing conditions, one primer sequence being specific to the 3′ end of the first strand of the nucleic acid molecule to be sequenced and the other specific to the 3′ end of the complementary strand;    d) ligating a double-stranded promoter sequence to the fragment of the nucleic acid molecule obtained in step (c);    e) determining the first terminal nucleotide sequence of the fragment of the nucleic acid molecule obtained in the step (d) by the method of  claim 19 , wherein the RNA polymerase is specific to the first primer sequence containing a phage promoter and the reverse transcriptase is primed with the promoter which was ligated in step (d) thereby determining the nucleotide sequence of the first terminal; and    f) determining the second terminal nucleotide sequence of the nucleic acid by the method of  claim 19 , wherein the polymerase is specific to the second primer sequence containing a promoter and the reverse transcriptase is primed with the promoter which was ligated in step (d) thereby determining the nucleotide sequence of the second terminal.    
     
     
         43 . A method of  claim 42 , wherein the treating of step (d) comprises the use of the Klenow fragment.  
     
     
         44 . A method of determining the nucleotide sequence of sequences present within a nucleic acid molecule which are adjacent to areas of known sequence which comprises: 
 a) cleaving the nucleic acid molecule adjacent to the sequences of interest under conditions so as to generate fragments of the nucleic acid molecule which contain the sequences of interest;    b) if the fragments of the nucleic acid molecule do not have blunt ends, treating the fragments of the nucleic acid molecule so as to generate blunt ends;    c) contacting the fragments containing the sequences of interest obtained in step (a) or (b) with an oligonucleotide containing two different promoter sequences adjacent to each other by blunt end ligation under conditions such that the promoter sequence binds adjacent to the sequence of interest and it is unlikely that the fragment will bind a promoter at both ends;    d) transcribing the fragments containing the sequences of interest and promoter sequence obtained in step (c) using a polymerase specific to the 5′ promoter sequence;    e) degrading or removing the fragments which were generated in steps (a) and (b);    f) synthesizing a nucleic acid sequence complementary to the first sequence to be determined using a downstream primer specific for the known sequence adjacent to the first sequence to be determined;    g) amplifying the amount of fragments containing the sequence to be determined using a downstream primer specific for the known sequence adjacent to the second sequence to be determined and an upstream primer specific for the second promoter sequence;    h) transcribing the fragments containing the sequence of interest using a polymerase specific to the second promoter sequence;    i) sequencing using a downstream primer specific for the third known sequence.    
     
     
         45 . A method of  claim 44 , wherein step (b) further comprises treating the blunt ends of the fragments with an endonuclease to generate one 3′ overhang which is resistant to blunt end ligation at that end.  
     
     
         46 . A method of  claim 44 , wherein in step (a) the cleaving comprises treating the nucleic acid molecule with a restriction endonuclease.  
     
     
         47 . A method of  claim 44 , wherein in step (a) the cleaving comprises shearing the nucleotide.  
     
     
         48 . A method of  claim 44 , wherein step (b) further comprises the removal of self-priming RNA.  
     
     
         49 . A method of  claim 44 , wherein the amplifying of step (g) comprises multiple rounds of polymerase chain reaction.  
     
     
         50 . A method of detecting a point mutation or polymorphism in a nucleic acid molecule which comprises: 
 a) amplifying the sequence of interest present within the nucleic acid molecule by the method of  claim 1  or  2 , wherein the oligonucleotide primer sequence of interest hybridizes to a sequence of the nucleic acid molecule containing the nucleotide point mutation;    b) determining the amount RNA produced in step (G) of  claim 1  or  2 ; and    c) comparing the amount of RNA corresponding to the sequence of interest which has been produced with the amount of RNA expected, an increased amount of RNA indicating the presence of point mutation.    
     
     
         51 . A method of carrier testing which comprises: 
 a) obtaining a sample containing the nucleic acid molecule of interest from a subject; and    b) detecting the presence of a point mutation in the nucleic acid molecule of interest using the method of  claim 50  thereby determining whether the subject is a carrier.    
     
     
         52 . A method of prenatal diagnosis which comprises: 
 a) obtaining a sample containing the nucleic acid molecule of interest from a subject; and    b) detecting the presence of a point mutation in the nucleic acid molecule of interest using the method of  claim 50  thereby determining whether the subject has the tested for mutation.    
     
     
         53 . A method of detecting the presence a mutation or polymorphism in a nucleic acid molecule which comprises: 
 a) amplifying the sequence of interest present within the nucleic acid molecule in a sample by the method of  claim 1  or  2 ;    b) separating the amplified sequence of interest generated in step (a) from the sample; and    c) comparing the sequence obtained in step (b) with a normal sequence thereby detecting the presence of a mutation or polymorphism.    
     
     
         54 . A method of determining the exonic nucleotide sequence of a gene which comprises determining the nucleotide sequence of the mRNA transcribed by the gene using the method of  claim 27  and deducing the complementary sequence of nucleotide, thereby determining the exonic sequence of the gene.  
     
     
         55 . A method of detecting mutations in RNA in tissues not accessible to direct analysis which comprises determining the exonic nucleotide sequence of a gene using the method of  claim 54 , and comparing the nucleotide sequence obtained with the normal nucleotide sequence, any difference in the sequence indicating a genomic mutation.  
     
     
         56 . A method of  claim 55 , wherein the gene is the Factor IX gene.  
     
     
         57 . A method of determining the predisposition of a subject to hemophilia B, which comprises determining the exonic sequence of the gene using the method of  claim 56  and comparing the nucleotide sequence so obtained with normal and known genetic mutants thereby determining the subject's predisposition to the disease.  
     
     
         58 . A method of sequencing homologous genes in different species which comprises determining the exonic sequence of the gene of interest using the method of  claim 27  wherein the gene of interest is identified by binding a primer corresponding to a nucleic acid sequence determined in a different species.  
     
     
         59 . A method of a sequencing a region of a nucleic acid molecule which is adjacent to a known region of a known sequence which comprises: 
 a) annealing an oligonucleotide containing a promoter to the known region of the nucleic acid molecule;    b) extending the oligonucleotide to the region to be sequenced so that the extension product for primer is complementary to the unknown region of the nucleic acid;    c) isolating the portion of the oligonucleotide extension product which is complementary to the region to be sequenced;    d) treating the oligonucleotide extension product which is complementary to the region to be sequenced so as to add a promoter;    e) transcribing the sequence of the oligonucleotide extension product;    f) treating the transcript so produced so as to prepare a cDNA which is complementary to the transcript; and    g) sequencing the cDNA using the method of  claim 27 .    
     
     
         60 . A method of detecting and determining mutations and polymorphisms in the sequence of a nucleic acid which comprises: 
 a) determining the sequence of the nucleic by the method of  claim 27;  and    b) comparing the sequence obtained with that of the normal sequence, known mutations, and polymorphisms.    
     
     
         61 . A method of  claim 60 , wherein the mutation is detected in an oncogene.  
     
     
         62 . A method of monitoring the progression of a cancer which comprises detecting and determining mutation and polymorphism in an oncogene using the method of  claim 61 , and comparing the types of mutation and polymorphism determined with the type of mutation and polymorphism determined at earlier points of time, a change in the types of mutation and polymorphism indicating the progression of the disease.  
     
     
         63 . A method of monitoring the efficiency of treatment of a cancer which comprises detecting and determining mutation and polymorphism in an oncogene using the method of  claim 61 , and comparing the type of mutation and polymorphism with the type of mutation and polymorphism determined at earlier points in time, a change in the types of mutation and polymorphism indicating the efficiency of the treatment.  
     
     
         64 . A method of diagnosing and subtyping infectious agents which comprises: 
 a) obtaining a sample containing the agent to be analyzed;    b) treating the sample so as to make the nucleic acid molecule to be tested accessible to analysis;    c) determining the nucleotide sequence of the nucleic acid molecule from the infectious agent by the method of  claim 60;  and    d) comparing the nucleotide sequence obtained with known sequences of nucleotide, thereby diagnosing and subtyping the infectious agents.