Amplification of nucleic acid sequences

What is claimed is:

1. A process for amplifying enzymatically a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids while reducing strand displacement by polymerase, comprising the steps of:

a) selecting the target nucleic acid sequence;

b) providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence and whose 3' end is adjacent to the 5' end of the first primer and a third primer which is substantially complementary to at least a portion of said first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which reduces strand displacement under polymerizing conditions;

c) providing at least four different nucleotide bases;

d) hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e) ligating under conditions such that the adjacent 5' end of the first primer and the 3' end of the second primer will ligate to form a fused amplification product substantially complementary to said target nucleic acid sequence;

f) dissociating said fused amplification product from said target nucleic acid sequence;

g) hybridizing said third primer to said fused amplification product;

h) extending said third primer in the presence of the nucleotide bases under conditions such that an extended amplification product is formed substantially complementary to said fused amplification product and which contains said modification;

i) dissociating the extended amplification product from the fused amplification product;

j) allowing the extended, modified amplification product to hybridize to additional first and second primers in a target dependent manner; and

k) ligating the 5' end of the additional first primer to the 3' end of the additional second primer while reducing strand displacement of the additional first primer, to form additional amplification product.

2. The process of claim 1, wherein the modification is to the nucleotide base.

3. The process of claim 2, wherein the modification is a base selected from the group consisting of xanthine, hypoxanthine and an abasic site.

4. The process of claim 1, wherein the modification is to the nucleotide sugar moiety.

5. The process of claim 4, wherein the modification is selected from the group consisting of 2'-O-alkylribonucleotides, ribonucleotides, and arabinosyl nucleotide derivatives.

6. The process of claim 5, wherein the modification is a 2'-O-methylribonucleotide.

7. The process of claim 1, wherein the modification is to the internucleotide linkage.

8. The process of claim 7, wherein the modification is a methyl phosphonate internucleotide linkage.

9. The process of claim 1, wherein the target nucleic acid is single stranded.

10. The process of claim 1, wherein steps (d) through (k) are repeated at least once.

11. The process of claim 1, wherein the target nucleic acid is DNA.

12. The process of claim 1, wherein the target nucleic acid is RNA.

13. The process of claim 1, wherein steps (e) and (k) are conducted in the presence of a ligating enzyme.

14. The process of claim 13, wherein the ligating enzyme is T4 DNA ligase.

15. The process of claim 13, wherein the ligating enzyme is stable at 0.degree.-95.degree. C.

16. The process of claim 15, wherein the ligating enzyme is selected from the group consisting of Taq ligase, Pfu ligase and Ampligase.

17. The process of claim 1, wherein step (h) is conducted in the presence of a polymerase.

18. The process of claim 17, wherein the polymerase is selected from the group consisting of E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, and T4 DNA polymerase.

19. The process of claim 17, wherein the polymerase is stable at temperatures of 0.degree.-95.degree. C.

20. The process of claim 19, wherein the polymerase is selected from the group consisting of Taq DNA polymerase, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, AmpliTaq DNA polymerase Stoffel fragment, T4 DNA polymerase, Hot Tub DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Pfu or Exo-Pfu DNA Polymerase, RNA polymerase and reverse transcriptase.

21. The process of claim 1, wherein the target nucleic acid sequence contains at least one deletion or mutation that causes a genetic disease or cancer.

22. The process of claim 1, wherein the target nucleic acid sequence is contained in a plant, animal, insect, pathogenic organism, virus or oncogene.

23. The process of claim 1, wherein one of said primers comprises two or more different oligonucleotides, one of said oligonucleotides having a sequence exactly complementary to said target nucleic acid sequence.

24. The process of claim 1, wherein each of the steps is conducted sequentially without isolation or purification of the products.

25. The process of claim 24 wherein each of the steps is conducted in a single reaction medium.

26. The process of claim 1, wherein the 5' end of the first primer comprises a phosphorothioate group.

27. The process of claim 1, wherein the 3' end of the second primer comprises an arabinosyl nucleotide.

28. A process for detecting enzymatically a mutation or an allele in a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, comprising the steps of:

a) selecting the target nucleic acid sequence;

b) providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence and whose 3' end is adjacent to the 5' end of the first primer and a third primer which is substantially complementary to at least a portion of said first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which reduces strand displacement under polymerizing conditions, wherein one of said primers comprises two or more different oligonucleotides, one of said oligonucleotides having a sequence exactly complementary to said target nucleic acid sequence wherein each oligonucleotide is labeled with a different label;

c) providing at least four different nucleotide bases;

d) hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e) ligating under conditions such that the adjacent 5' end of the first primer and the 3' end of the second primer will ligate to form a fused amplification product substantially complementary to said target nucleic acid sequence;

f) dissociating said fused amplification product from said target nucleic acid sequence;

g) hybridizing said third primer to said fused amplification product;

h) extending said third primer in the presence of the nucleotide bases under conditions such that an extended amplification product is formed substantially complementary to said fused amplification product and which contains said modification;

i) dissociating the extended, modified amplification product from the fused amplification product;

j) allowing the extended, modified amplification product to hybridize to additional first and second primers in a target dependent manner; and

k) ligating the 5' end of the additional first primer to the 3' end of the additional second primer while reducing strand displacement of the additional first primer, to form additional amplification product; and

l) determining which labeled primer is contained within the fused amplification product or the extended amplification product to thereby detect whether the mutation or allele is present.

29. A process for amplifying enzymatically a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising the steps of:

a) selecting the target nucleic acid sequence;

b) providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence said second segment being spaced a number of nucleotides from said first segment, and a third primer which is substantially complementary to at least a portion of said first primer, said portion including the 5' end of the first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which reduces strand displacement under polymerizing conditions;

c) providing at least four different nucleotide bases;

d) hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e) extending the 3' end of the second primer in the presence of the nucleotide bases under conditions such that an extended second primer is formed wherein the 3' end of the extended second primer terminates at a base adjacent to the 5' end of the first primer;

f) ligating the ends of the first primer and extended second primer under conditions such that said first and said second primers will form a fused amplification product substantially complementary to said target nucleic acid sequence;

g) dissociating said fused amplification product from said target nucleic acid sequence;

h) hybridizing said third primer to said fused amplification product;

i) extending said third primer in the presence of the nucleotide bases under conditions such that an extended modified amplification product is formed substantially complementary to said fused amplification product and which contains said modification;

j) allowing the extended, modified amplification product to hybridize to additional first and second primers in a target dependent manner;

k) extending the 3' end of the additional second primer in the presence of the nucleotide bases under conditions such that an extended additional second primer is formed wherein the 3' end of the extended additional second primer terminates at a base adjacent to the 5' end of the additional first primer; and

l) ligating the 5' end of the additional first primer to the 3' end of the additional second primer while reducing strand displacement of the additional first primer, to form additional amplification product.

30. The process of claim 29, wherein the modification is to the nucleotide base.

31. The process of claim 30, wherein the modification is a base selected from the group consisting of xanthine, hypoxanthine and an abasic site.

32. The process of claim 29, wherein the modification is to the nucleotide sugar moiety.

33. The process of claim 32, wherein the modification is selected from the group consisting of 2'-O-alkylribonucleotides, ribonucleotides, and arabinosyl nucleotide derivatives.

34. The process of claim 33, wherein the modification is a 2'-O-methylribonucleotide.

35. The process of claim 29, wherein the modification is to the internucleotide linkage.

36. The process of claim 35, wherein the modification is a methyl phosphonate internucleotide linkage.

37. The process of claim 29, wherein steps (d) through (j) are repeated at least once.

38. The process of claim 29, wherein the target nucleic acid is double stranded nucleic acid comprising a first and second strand wherein said first and second primers are substantially complementary to said first strand and said third primer is substantially complementary to said second strand and said first and second strands are dissociated prior to step (d), and wherein at least some of the third primers hybridize to the second strand and are extended to form an extended amplification product.

39. The process of claim 38, further comprising a fourth primer wherein the fourth primer is substantially complementary to said second target nucleic acid strand and said fourth primer is substantially complementary to said second primer, and wherein the third primer is extended to the 3' end of the fourth primer and is ligated thereto.

40. The process of claim 39, wherein the nucleic acid is denatured by heating.

41. The process of claim 29, wherein the nucleic acid is DNA from a plant, animal, insect, pathogenic organism, virus or oncogene.

42. The process of claim 29, wherein the nucleic acid is RNA.

43. The process of claim 29, wherein steps (f) and (l) are conducted in the presence of a ligating enzyme.

44. The process of claim 43, wherein the ligating enzyme is T4 DNA ligase.

45. The process of claim 43, wherein the ligating enzyme is stable at 0.degree.-95.degree. C.

46. The process of claim 45, wherein the ligating enzyme is selected from the group consisting of Ampligase, Taq ligase and Pfu ligase.

47. The process of claim 29 wherein steps (e), (i) and (k) are conducted in the presence of polymerase.

48. The process of claim 47, wherein the polymerase is selected from the group consisting of E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I and T4 DNA polymerase.

49. The process of claim 47, wherein the polymerase is stable at 0.degree.-95.degree. C.

50. The process of claim 49, wherein the polymerase is selected from the group consisting of Taq DNA polymerase, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, AmpliTaq DNA polymerase Stoffel fragment, T4 DNA polymerase, Hot Tub DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Pfu or Exo-Pfu DNA Polymerase, RNA polymerase and reverse transcriptase.

51. The process of claim 29, wherein the target nucleic acid sequence contains at least one deletion or mutation that causes a genetic disease.

52. The process of claim 29, wherein the target nucleic acid sequence is contained in a pathogenic organism, virus or oncogene.

53. The process of claim 29, wherein one of said primers comprises two or more oligonucleotides, one of said oligonucleotides having a sequence exactly complementary to said target nucleic acid.

54. The process of claim 29, wherein each of the steps is conducted sequentially without isolation or purification of the products.

55. The process of claim 54, wherein each of the steps is conducted in a single reaction medium.

56. The process of claim 29, wherein the 5' end of the first primer comprises an phosphorothioate group.

57. The process of claim 39, wherein the 5' end of the fourth primer comprises an phosphorothioate group.

58. A process for detecting enzymatically a mutation or an allele in a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising the steps of

a) selecting the target nucleic acid sequence;

b) providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence said second segment being spaced from said first segment and a third primer which is substantially complementary to at least a portion of said first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which reduces strand displacement under polymerizing conditions, wherein one of said primers comprises two or more different oligonucleotides, one of said oligonucleotides having a sequence exactly complementary to said target nucleic acid sequence wherein each oligonucleotide is labeled with a different label;

c) providing at least four different nucleotide bases;

d) hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e) extending the 3' end of the second primer in the presence of the nucleotide bases under conditions such that an extended second primer is formed wherein the 3' end of the extended second primer is adjacent to the 5' end of the first primer;

f) ligating the ends of the first and second primers under conditions such that said first and said extended second primers will form a fused amplification product complementary to said target nucleic acid sequence;

g) dissociating said fused amplification product from said target nucleic acid sequence;

h) hybridizing said third primer to said fused amplification product;

i) extending said third primer in the presence of the nucleotide bases under conditions such that an extended amplification product is formed complementary to said fused amplification product and which contains said modification;

j) optionally dissociating the extended amplification product from the fused amplification product;

k) determining which labeled primer is contained within the fused amplification product or the extended amplification product to thereby detect whether the mutation or allele is present.

59. A process according to any of claims 1, 28, 29 or 58 wherein the first primer hybridizes to the target sequence prior to the second primer.

Description Submit all comments and votes

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a process for amplifying nucleic acid sequences. More specifically, it relates to an improved process for producing nucleic acid sequences from a DNA or RNA template which may be purified, or may exist in a mixture of nucleic acids. The resulting nucleic acid sequences may be exact copies of the template, or may be modified.

2. Description of Related Art

In the past, methods have been employed for amplifying nucleic acid sequences wherein both strands of the nucleic acid sequence to be amplified are synthesized by the same method. Such methods are prone to limitations due to the nature of the enzymes utilized in these processes.

In U.S. Pat. Nos. 4,683,195 and 4,683,202, DNA or RNA is amplified by the polymerase chain reaction (PCR). These patents are incorporated herein by reference in their entirety. This method involves the hybridization of an oligonucleotide primer to the 5' end of each complementary strand of the double-stranded target nucleic acid. The primers are extended from the 3' end in a 5'.fwdarw.3' direction by a DNA polymerase which incorporates free nucleotides into a nucleic acid sequence complementary to each strand of the target nucleic acid. After dissociation of the extension products from the target nucleic acid strands, the extension products become target sequences for the next cycle. In order to obtain satisfactory amounts of the amplified DNA, repeated cycles must be carried out, between which cycles, the complementary DNA strands must be denatured under elevated temperatures.

Traditional polymerases used in this process, such as E. coli DNA polymerase I have the limitation of being inactivated at temperatures necessary for the denaturation of the complementary strands. Thus, between each cycle of synthesis by such polymerases and after the heat denaturation step, a fresh aliquot of enzyme must be added to the reaction mixture so that extension of the primer and synthesis of the complementary strand may occur in the following cycle. This additional step increases the time required for amplification and decreases the ease of amplification which requires multiple steps.

In recent years, thermostable DNA polymerases have been discovered and isolated from thermophilic organisms such as Thermus aquaticus. Such thermostable polymerases make it possible to add enzyme at the beginning of a series of synthesis and denaturation steps, without the need to add a new aliquot of enzyme after each denaturation step.

A potential problem associated with PCR is the hybridization of a primer sequence to regions of the DNA molecule not intended to be amplified. Generally these undesired hybridizations occur because the target sample contains, in addition to the target sequence itself, other sequences with some complementarity to the primer sequences. If the 3' terminal nucleotides of the primer molecule are successfully hybridized to a sequence other than the target sequence, it is possible that primer extension may be successfully initiated by the polymerase enzyme, leading to the generation of an extension product different from the desired target sequence. Under some circumstances, this extension product will undergo exponential amplification, and be erroneously thought to be the desired target sequence.

A method of detecting a specific nucleic acid sequence present in low copy in a mixture of nucleic acids, called ligase chain reaction (LCR), has also been described. European patent application 0 320 308 describes this method and is incorporated herein by reference in its entirety. Target nucleic acid in a sample is annealed to probes containing contiguous sequences. Upon hybridization, the probes are ligated to form detectable fused probes complementary to the original target nucleic acid. The fused probes are disassociated from the nucleic acid and serve as a template for further hybridizations and fusions of the probes, thus amplifying geometrically the nucleic acid to be detected. The method does not use DNA polymerase.

LCR has disadvantages due to the need for at least four separate oligonucleotide probes for amplification. It also requires that the entire sequence of the target nucleic acid be known. Further, background signal can be caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, when added in excess, can easily form duplexes among themselves which can be ligated independently of the target nucleic acid.

European Application No. 0 439 182 which is incorporated herein in its entirety by reference discloses a method of improving LCR amplification by providing probes/primers which hybridize to the target nucleic acid wherein one end is modified such that ligation cannot occur until the modified end is corrected. One such modification is the placement of a small gap between the probes preventing ligation of the probes. The gap sequence of the target nucleic acid must be selected such that the DNA sequence is comprised of three or less different nucleotides from the four possible nucleotides. The fourth nucleotide must be the first base complementary to the 5' end of the adjacent probe. The gap is then filled using a DNA polymerase or reverse transcriptase to extend one or more of the probes in a 5' to 3' direction in a target dependent manner to render the probes ligatable. The reaction mixture omits the fourth deoxynucleoside triphosphate complementary to the base at the 5' end of the adjacent probe. Because this method requires that the gap chosen in the target nucleic acid only contains bases which are complementary to a maximum of three of the deoxynucleoside triphosphates, the method limits the location of the gap on the target nucleic acid and also limits the size of the gap. Further, the method requires four primers. The application also discloses a method of PCR amplification wherein one end of the primer is modified such that the primer is not extendable by a polymerase enzyme. When this modification is removed in a template dependent manner, the primer can be extended. However, this type of PCR requires an additional step of removal of the modification before extension can occur.

In view of the foregoing disadvantages attendant with prior art methods of amplifying nucleic acid sequences, it should be apparent that there exists a need in the art for a method in which the fidelity of amplified sequences can be increased, which allows for the detection of a particular nucleic acid strand, and which allows one to efficiently examine multiple alleles in a single series of amplification steps.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that certain aspects of LCR and PCR can be used in combination to detect and amplify a target nucleic acid sequence with increased fidelity. Accordingly, in one of its process aspects, the present invention relates to a process for amplifying enzymatically a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids while substantially avoiding strand displacement by polymerase, comprising the steps of:

a. selecting the target nucleic acid sequence;

b. providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence and whose 3' end is adjacent to the 5' end of the first primer and a third primer which is similar to the first end of the target nucleic acid sequence and which is substantially complementary to at least a portion of said first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which substantially avoids strand displacement under polymerizing conditions;

c. providing at least four different nucleotide bases;

d. hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e. ligating under conditions such that the adjacent 5' end of the first primer and the 3' end of the second primer will ligate to form a fused amplification product substantially complementary to said target nucleic acid sequence;

f. dissociating said fused amplification product from said target nucleic acid sequence;

g. hybridizing said third primer to said fused amplification product;

h. extending said third primer in the presence of the nucleotide bases under conditions such that an extended amplification product is formed substantially complementary to said fused amplification product and which contains said modification;

i. dissociating the extended amplification product from the fused amplification product;

j. allowing the extended, modified amplification product to hybridize to additional first and second primers in a target dependent manner; and

k. ligating the 5' end of the additional first primer to the 3' end of the additional second primer while substantially avoiding strand displacement of the additional first primer, to form additional amplification product.

In another of its process aspects, the present invention relates to a process for detecting enzymatically a point mutation or allele of a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids using the method disclosed above. One of said primers is comprised of a number of similar oligonucleotide sequences, one of which is exactly complementary to the possible allele or point mutation and each of which oligonucleotides is labeled with a different label. The allele is determined by detecting which labeled oligonucleotide is contained within the resulting amplification products.

In a third aspect, the present invention relates to a process for amplifying enzymatically a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising the steps of:

a. selecting the target nucleic acid sequence;

b. providing primers, said primers comprising a first primer which is substantially complementary to a first segment at a first end of the target nucleic acid sequence and a second primer which is substantially complementary to a second segment at a second end of the target nucleic acid sequence said second segment being spaced a number of nucleotides from said first segment and a third primer which is similar to the first end of the target nucleic acid sequence and which is substantially complementary to at least a portion of said first primer, said portion including the 5' end of the first primer such that when the third primer is hybridized to the first primer, the position of the third primer complementary to the base at the 5' end of the first primer contains a modification which substantially avoids strand displacement under polymerizing conditions;

c. providing at least four different nucleotide bases;

d. hybridizing said first and second primers to the target nucleic acid sequence in a target dependent manner to form a primer-target complex;

e. extending the 3' end of the second primer in the presence of the nucleotide bases under conditions such that an extended second primer is formed wherein the 3' end of the extended second primer terminates at a base adjacent to the 5' end of the first primer;

f. ligating the ends of the first primer and extended second primer under conditions such that said first and said second primers will form a fused amplification product substantially complementary to said target nucleic acid sequence;

g. dissociating said fused amplification product from said target nucleic acid sequence;

h. hybridizing said third primer to said fused amplification product;

i. extending said third primer in the presence of the nucleotide bases under conditions such that an extended modified amplification product is formed substantially complementary to said fused amplification product and which contains said modification;

j. allowing the extended, modified amplification product to hybridize to additional first and second primers in a target dependent manner;

k. extending the 3' end of the additional second primer in the presence of the nucleotide bases under conditions such that an extended additional second primer is formed wherein the 3' end of the extended additional second primer terminates at a base adjacent to the 5' end of the additional first primer; and

l. ligating the 5' end of the additional first primer to the 3' end of the additional second primer while substantially avoiding strand displacement of the additional first primer, to form additional amplification product.

In one of its product aspects, the present invention relates to a kit for amplifying a target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids comprising: first, second, and third primers and optionally a fourth primer as described above; a ligating enzyme; a polymerizing enzyme; and at least four nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts one embodiment of the method of DNA amplification/detection as set forth herein.

FIG. 2 is a printout from a Phosphor Imager of a scanned acrylamide gel. The arrow indicates the resulting higher molecular weight amplification products.

FIG. 3 depicts another embodiment of the method of DNA amplification/detection as set forth herein.

FIG. 4 shows a portion of the sequence of the multidrug resistance gene (MDR-1) (SEQ ID NO:1).

FIGS. 5-11 are printouts from a Phosphor Imager of a scanned acrylamide gel which show amplification achieved with various embodiments of the present invention.

FIG. 12 depicts another embodiment of the method of DNA amplification/detection as set forth herein.

FIGS. 13-14 depict other embodiments of the method of DNA amplification/detection as set forth herein.

FIG. 15 is a printout from a Phosphor Imager of a scanned acrylamide gel which shows amplification achieved with various embodiments of the present invention.

FIG. 16 depicts an experiment which shows that a template modification according to the present invention does not have a significant effect on DNA ligase activity.

FIG. 17 is a printout from a Phosphor Imager of a scanned acrylamide gel which shows results obtained from the experiment depicted in FIG. 16.

FIG. 18 is a printout from a Phosphor Imager of a scanned acrylamide gel which shows results obtained from the experiment described in Example 14.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Prior to discussing this invention in detail, the following terms will first be defined:

The "target nucleic acid" or "target nucleic acid sequence" suitable for use in the present invention may be taken from prokaryotic or eukaryotic DNA or RNA, including from plants, animals, insects, microorganisms, etc., and it may be isolated or present in samples which contain nucleic acid sequences in addition to the target nucleic acid sequence to be amplified. The target nucleic acid sequence may be located on a nucleic acid strand which is longer than the target nucleic acid sequence. In this case, the ends of the target nucleic acid sequence are the boundaries with the unselected nucleic acid sequence and the target nucleic acid sequence. The target nucleic acid sample may be obtained synthetically, or can be isolated from any organism by methods well known in the art. Particularly useful sources of nucleic acid are derived from tissues or blood samples of an organism, nucleic acids which are present in self-replicating vectors, and nucleic acids derived from viruses and pathogenic organisms such as bacteria and fungi. Also particularly useful for the present invention are target nucleic acid sequences which are related to disease states, such as those caused by chromosomal rearrangement, insertions, deletions, translocations and other mutations, those caused by oncogenes, and those associated with cancer.

The term "selected" means that a target nucleic acid sequence having the desired characteristics is located and probes are constructed around appropriate segments of the target sequence.

The term "probe" or "primer" has the same meaning herein, namely, an oligonucleotide fragment which is single stranded. The term "oligonucleotide" means DNA or RNA.

A probe or primer is "substantially complementary" to the target nucleic acid sequence if it hybridizes to the sequence under renaturation conditions so as to allow target dependent ligation or extension. Renaturation depends on specific base pairing between A-X (where X is T or U) and G-C bases to form a double stranded duplex structure. Therefore, the primer sequence need not reflect the exact sequence of the target nucleic acid sequence. However, if an exact copy of the target nucleic acid is desired, the primer should reflect the exact sequence. Typically, a "substantially complementary" primer will contain at least 70% or more bases which are complementary to the target nucleic segment. More preferably 80% of the bases are complementary and most preferably 90% of the bases are complementary. Generally, the primer must hybridize to the target nucleic acid sequence at the end to be ligated or extended to allow target dependent ligation or extension.

The primers may be RNA or DNA, and may contain modified nitrogenous bases which are analogs of the normally incorporated bases, or which have been modified by attaching labels or linker arms suitable for attaching labels. Inosine may be used at positions where the target sequence is not known, or where it may be degenerate. The oligonucleotides must be sufficiently long to allow hybridization of the primer to the target nucleic acid and to allow amplification to proceed. They are preferably 15 to 50 nucleotides long, more preferably 20 to 40 nucleotides long, and most preferably 25 to 35 nucleotides long. The nucleotide sequence, content and length will vary depending on the sequence to be amplified.

It is contemplated that a primer may comprise one or more oligonucleotides which comprise substantially complementary sequences to the target nucleic acid sequence. Thus, under less stringent conditions, each of the oligonucleotides would hybridize to the same segment of the target nucleic acid. However, under increasingly stringent hybridization conditions, only that oligonucleotide sequence which is most complementary to the target nucleic acid sequence will hybridize. The stringency of conditions is generally known to those in the art to be dependant on temperature, solvent and other parameters. Perhaps the most easily controlled of these parameters is temperature and since the conditions here are similar to those of PCR, one skilled in the art could determine the appropriate conditions required to achieve the level of stringency desired.

Oligonucleotide primers or oligonucleotide probes suitable for use in the present invention may be derived by any method known in the art, including chemical synthesis, or by cleavage of a larger nucleic acid using non-specific nucleic acid-cleaving chemicals or enzymes, or by using site-specific restriction endonucleases.

The primers may be prepared using the .beta.-cyanoethylphosphoramidite method or other methods known in the art. A preferable method for synthesizing oligonucleotide primers is conducted using an automated DNA synthesizer by methods known in the art. Once the desired oligonucleotide primer is synthesized, it is cleaved from the solid support on which it was synthesized, and treated, by methods known in the art, to remove any protecting groups present. The oligonucleotide primer may then be purified by any method known in the art, including extraction and gel purification. The concentration and purity of the oligonucleotide primer may be examined on an acrylamide gel, or by measuring the optical densities at 260 and 280 nm in a spectrophotometer.

In order for the ligase to ligate the oligonucleotide primers, the primers used in the present invention are preferably phosphorylated at their 5' ends. This may be achieved by any method known in the art, but is preferably conducted with the enzyme T4 polynucleotide kinase. The oligonucleotides can be phosphorylated in the presence of unlabeled or labeled ATP. In order to monitor the amplification process, labeled ATP may be used to phosphorylate the primers. Particularly preferable is [.gamma.-.sup.32 P] ATP.

The oligonucleotide primers may alternatively be labeled with any detectable marker known in the art, including other radioactive nuclides such as .sup.35 S or .sup.3 H and the like, fluorescent markers such as fluorescein, rhodamine, Texas red, Lucifer yellow, AMCA blue and the like, or with enzymatic markers which may produce detectable signals when a particular chemical reaction is conducted, such as alkaline phosphatase or horseradish peroxidase. Such enzymatic markers are preferably heat stable, so as to survive the denaturation steps of the amplification process. Primers may be indirectly labeled by incorporating a nucleotide covalently linked to a hapten or other molecule such as biotin to which a labeled avidin molecule may be bound, or digoxygenin, to which a labeled anti-digoxygenin antibody may be bound.

Primers may be labeled during chemical synthesis or the label may be attached after synthesis by methods known in the art. The method of labeling and the type of label is not critical to this invention.

It is contemplated that the probes or primers may be modified. For example the hydrolysis of a primer by 5' to 3' exonuclease associated with polymerase may be prevented by placing a phosphorothioate group between the last nucleotides of the 5' end of the primer. The extension of a primer by polymerase can be blocked by placing a dideoxynucleotide, an amino group, a cordycepin, or a phosphate group at the 3' end. Alternatively, the extension of a primer may be blocked by placing an arabinosyl nucleotide at the 3' end of the primer which blocks extension by polymerase but allows ligation of the primer to another primer.

The term "the four different nucleotide bases" shall refer to deoxythymidine triphosphate (dTTP); deoxyadenosine triphosphate (dATP); deoxyguanosine triphosphate (dGTP); and deoxycytidine triphosphate (dCTP), when the context is DNA, but shall refer to uridine triphosphate (UTP); adenosine triphosphate (ATP); guanosine triphosphate (GTP); and cytidine triphosphate (CTP) when the context is RNA. Alternatively, dUTP, dITP, rITP or any other modified base may replace one of the four nucleotide bases or may be included along with the four nucleotide bases in the reaction mixture so as to be incorporated into the amplified strand. The amplification steps are conducted in the presence of at least the four deoxynucleoside triphosphates (dATP, dCTP, dGTP and dTTP) or a modified nucleoside triphosphate to produce a DNA strand, or in the presence of the four ribonucleoside triphosphates (ATP, CTP, GTP and UTP) or a modified nucleoside triphosphate to produce an RNA strand from extension of the oligonucleotide which acts as a primer.

Where the presence of a particular mutation or allele is to be detected by the methods of this invention, one of the oligonucleotide primers may comprise a set of oligonucleotide fragments, each differing in sequence and each labeled by a different method. That oligonucleotide fragment which is exactly complementary to the target DNA sequence will be detected by the presence of that label in the amplification products. In this case, each oligonucleotide fragment may be labeled as described above.

Utility

First Embodiment

In a first embodiment, the target nucleic acid is described as single stranded. However, this should be understood to include the case where the target is actually double stranded but is simply separated from its complementary strand prior to hybridization with probes/primers. Primers one and two, together, are substantially complementary to the target nucleic acid sequence and hybridize to adjacent regions of the target nucleic acid strand such that upon hybridization of the two primers to the target nucleic acid strand the 5' end of the first primer is adjacent to the 3' end of the second primer. The 3' end of the first primer is substantially complementary to the 5' end of the target nucleic acid sequence and the 5' end of the second primer is substantially complementary to the 3' end of the target nucleic acid sequence. The 5' end of the first primer is ligated to the 3' end of the second primer using ligase to create a fused amplification product in a double stranded complex. The fused primer is dissociated from the target nucleic acid.

The third primer is substantially complementary to all or at least a portion of the first primer and is similar to the 5' end of the target nucleic acid. The third primer should be complementary to enough of the first primer so that specific hybridization is achieved under the conditions used. The third primer may be smaller than the first primer or it may be larger than the first primer and also be substantially complementary to a portion of the second primer. The third primer is hybridized to the fused amplification product and extended by polymerase in the presence of at least four different nucleotide bases to form an extended amplification product which is substantially complementary to the fused amplification product. This comprises the first cycle.

Subsequently the double stranded complexes are dissociated. The oligonucleotide primers (1 and 2) are hybridized to the target nucleic acid sequence and the extended amplification product from the first cycle. Primer 3 is hybridized to the fused amplification product. Extension and ligation occur as before and the process can be repeated.

It is contemplated that the 3' end of the second primer may be modified to block the extension of the second primer by polymerase while still allowing ligation of the 3' end of the second primer to the 5' end of the first primer. Such modification may be, for example, the placement of an arabinosyl nucleotide at the 3' end of the second primer. Methods for the chemical synthesis of DNA oligomers containing cytosine arabinoside are known in the art (Beardsley, Nucl. Acid. Res. (1988) 16:9