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Claims  |
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What is claimed is:
1. A process for amplifying at least one specific DNA sequence contained in
a DNA or a mixture of nucleic acids, wherein if the DNA is
double-stranded, it consists of two separated complementary strands of
equal or unequal length, which process comprises:
(a) contacting the DNA with four different nucleoside triphosphates and two
oligonucleotide primers, for each different specific sequence being
amplified, wherein each primer is selected to be sufficiently
complementary to different strands of each specific sequence to hybridize
therewith, such that the extension product synthesized from one primer,
when separated from its complement, can serve as a template for synthesis
of the extension product of the other primer, at a temperature which
promotes hybridization of each primer to its complementary strand;
(b) contacting each strand, at the same time as or after step (a), with
thermostable enzyme which catalyzes combination of the nucleoside
triphosphates to form primer extension products complementary to each
strand of DNA;
(c) maintaining the mixture from step (b) at an effective temperature for
an effective time to promote the activity of the enzyme, and to
synthesize, for each different sequence being amplified, an extension
product of each primer which is complementary to each strand, but not so
high as to separate each extension product from its complementary strand;
(d) heating the mixture from step (c) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(e) cooling the mixture from step (d) to an effective temperature to
promote hybridization of each primer to each of the single-stranded
molecules produced in step (d); and
(f) maintaining the mixture from step (e) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand produced in step (d), but not
so high as to separate each extension product from its complementary
strand, wherein steps (e) and (f) are conducted simultaneously or
sequentially.
2. The process of claim 1, wherein one specific DNA acid sequence is
amplified and two primers are employed.
3. The process of claim 1, wherein after steps (d)-(f) are repeated at
least once, a second set of two primers is added, wherein the primers
added are sufficiently complementary to different strands at internal
sequences of the amplified sequence to hybridize therewith, such that the
extension product synthesized from one primer, when separated from its
complement, can serve as a template for synthesis of the extension product
of the other primer.
4. The process of claim 1, wherein steps (d), (e) and (f) are repeated at
least five times.
5. The process of claim 4, wherein aid thermostable enzyme is a polymerase
from Thermus aquaticus.
6. The process of claim 3 wherein in step (a) the triphosphates and
primer(s) are contained in a buffer comprising 1.5-2 mM of a magnesium
salt, 150-200 .mu.M each of the triphosphates, and 1 .mu.M 58.degree. C.,
and step (d) is carried out at about 90-100.degree. C.
7. The process of claim 1, wherein said primers are
oligodeoxyribonucleotides.
8. The process of claim 1, wherein said DNA is cDNA.
9. The process of claim 1, wherein the heating and cooling steps (d)-(f)
are automated by a machine which controls temperature levels, transitions
from one temperature to another, and the timing of temperature levels.
10. The process of claim 1, wherein each primer is present in a molar ratio
of at least 1000:1 primer:complementary strand.
11. The process of claim 1, wherein at least one primer contains at least
one nucleotide which is not complementary to the specific sequence to be
amplified.
12. The process of claim 11, wherein said primer that contains at least one
nucleotide which is not complementary to the specific sequence to be
amplified encodes a promoter.
13. The process of claim 1, wherein steps (e) and (f) are carried out
sequentially, steps (c) and (f) take place at 40-80.degree. C., step (d)
takes place at 90-105.degree. C., and step (e) takes place at
35-65.degree. C.
14. The process of claim 1 wherein steps (e) and (f) are carried out
sequentially, steps (c) and (f) take place at 50-75.degree. C., step (d)
takes place at 90-100.degree. C., and step (e) takes place at 37.degree.
C.-60.degree. C.
15. The process of claim 1 wherein steps (e) and (f) are carried out
simultaneously at about 45-70.degree. C.
16. The process of claim 1, wherein said specific DNA sequence is contained
in a mixture of nucleic acids.
17. The process of claim 16, wherein said specific DNA sequence is
contained in a larger sequence.
18. The process of claim 1, wherein said specific DNA sequence is contained
in a larger sequence.
19. A process for detecting the presence of a specific DNA sequence in a
sample, said process comprising:
(a) amplifying said sequence by the process of claim 1; and
(b) determining if amplification has occurred.
20. A process for amplifying at least one specific DNA sequenc contained in
a DNA or a mixture of nucleic acids, wherein the DNA consists of two
complementary strands of equal or unequal length, which process comprises:
(a) heating the DNA in the presence of four different nucleoside
triphosphates and two oligonucleotide primers, for each different specific
sequencing being amplified, for an effective time and at an effective
temperature to denature each nucleic acid, wherein each primer is selected
to be sufficiently complementary to different strands of each specific
sequence to hybridize therewith, such that the extension product
synthesized from one primer, when separated from its complement, can serve
as a template for synthesis of the extension product of the other primer;
(b) cooling the denatured DNA to temperature which promotes hybridization
of each primer to its complementary strand;
(c) contacting the denatured DNA, at the same time as or after step (a) or
(b), with a thermostable enzyme which enables combination of the
nucleoside triphosphates to form primer extension products complementary
to each strand of DNA;
(d) maintaining the mixture from step (c) at an effective temperature for
an effective time to promote the activity of the thermostable enzyme, and
to synthesize, for each different sequence being amplified, an extension
product of each primer which si complementary to each strand, but not so
high as to separate each extension product from its complementary strand;
(e) heating the mixture from step (d) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(f) cooling the mixture from step (e) for an effective time and to an
effective temperature to promote hybridization of the primer to its
complementary single-stranded molecule produced in step (e); and
(g) maintaining the mixture from step (f) at an effective temperature for
an effective time to promote the activity of the enzyme, and to
synthesize, for each different sequence being amplified, an extension
product of each primer which is complementary to each strand produced in
step (f), but not so high as to separate each extension product form its
complementary strand, wherein steps (f) and (g) are carried out
simultaneously or sequentially.
21. The process of claim 20, wherein said specific DNA sequence is
contained in a mixture of nucleic acids.
22. The process of claim 21, wherein said specific DNA sequence is
contained in a
23. The process of claim 20, wherein said specific DNA sequence is
contained in a larger sequence.
24. A process for detecting the presence of a specific DNA sequence in a
sample, said process comprising:
(a) amplifying said sequence by the process of claim 20, and
(b) determining if amplification has occurred.
25. A process for detecting the presence or absence of at least one
specific DNA sequence in a sample containing a DNA or mixture of nucleic
acids, or distinguishing between two different DNA sequences in said
sample, wherein the sample is suspected of containing said sequence or
sequences, and wherein if the nucleic acid(s) are double-stranded, they
each consist of two separated complementary strands of equal or unequal
length, which process comprises:
(a) contacting the sample with four different nucleoside triphosphates and
two oligonucleotide primers, for each different specific sequence being
detected, wherein each primer is selected to be sufficiently complementary
to different strands of each specific sequence to hybridize therewith,
such that the extension product synthesized from one primer, when
separated from its complement, can serve as a template for synthesis of
the extension product of the other primer, at a temperature which promotes
hybridization of each primer to its complementary strand;
(b) contacting the sample, at the same time as or after step (a), with a
thermostable enzyme which catalyzes combination of the nucleoside
triphosphates to form primer extension products complementary to each
strand of DNA;
(c) maintaining the mixture from step (b) at an effective temperature for
an effective time to promote the activity of the enzyme, and to
synthesize, for each different sequence being detected, an extension
product of each primer which is complementary to each strand, but not so
high as to separate each extension product from its complementary strand;
(d) heating the mixture from step (c) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized to produce single-stranded
molecules, but not so high as to denature irreversibly the thermostable
enzyme;
(e) cooling the mixture from step (d) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced in step (d);
(f) maintaining the mixture from step (e) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being detected, but not so high as to separate
each extension product from its complementary strand, resulting in
amplification in quantity of the specific nucleic acid sequence or
sequences if present, wherein steps (e) and (f) are carried out
simultaneously or sequentially;
(g) adding to the product of step (f) a labeled oligonucleotide probe for
each sequence being detected capable of hybridizing to said sequence or to
a mutation thereof; and
(h) determining whether said hybridization has occurred.
26. A process for detecting the presence or absence of at least one
specific DNA sequence in a sample containing DNA or mixture of nucleic
acids, or distinguishing between two different DNA sequences in said
sample, where the sample is suspected of containing said sequence or
sequences and the DNA is double-stranded, which process comprises:
(a) heating the sample in the presence of four different nucleoside
triphosphates and two oligonucleotide primers, for each different specific
sequence being detected, for an effective time and at an effective
temperature to denature the DNA in the sample, wherein each primer is
selected to be sufficiently complementary to different strands of each
specific sequence to hybridize therewith, such that the extension product
synthesized from one primer, when separated from its complement, can serve
as a template for synthesis of the extension product of the other primer,
(b) cooling the denatured DNA to a temperature promotes hybridization of
each primer to its complementary strand;
(c) contacting the natured DNA, at the same time as or after step (a) or
(b), with a thermostable enzyme which catalyzes combination of the
nucleoside triphosphates to form primer extension products complementary
to each strand of DNA;
(d) maintaining the mixture from step (c) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being detected, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand;
(e) heating the mixture from step (d) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(f) cooling the mixture from step (e) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced from step (e);
(g) maintaining the mixture from step (f) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being detected, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand, resulting
in amplification in quantity of the specific nucleic acid sequence or
sequences if present, wherein steps (f) and (g) are carried out
simultaneously or sequentially;
(h) adding to the product of step (g) a labeled oligonucleotide probe for
each sequence being detected capable of hybridizing to said sequence or to
a mutuation thereof; and
(i) determining whether said hybridizing has occurred. determining whether
said hybridization has occurred.
27. The process of claim 26, wherein one specific DNA sequence is being
detected and two primers are employed, and before step (a) the DNA is
extracted from the sample.
28. The process of claim 26, wherein steps (d), (e) and (f) are repeated at
least 20 times.
29. The process of claim 26, wherein said thermostable enzyme is a
polymerase from Thermus aquaticus.
30. The process of claim 26, wherein said primers are
oligodeoxyribonucleotides.
31. The process of claim 26, wherein said DNA is cDNA.
32. The process of claim 26, wherein the heating and cooling steps (e)-(g)
are automated by a machine which controls temperature levels, transitions
from one temperature to another, and the timing of temperature levels.
33. The process of claim 26, wherein the specific DNA sequence to be
detected can cause a genetic, infectious or cancerous disease.
34. The process of claim 33, wherein the genetic disease is sickle cell
anemia or hemoglobin C disease.
35. The process of claim 34, wherein after step (g) and before step (h) the
sample is cut with a restriction enzyme and electrophoresed, and step (i)
is accomplished by Southern blot analysis.
36. A process for cloning into a cloning vector one or more specific DNA
sequences contained in a DNA or a mixture of nucleic acids, which DNA when
double-stranded consists of two separated complementary strands, and which
DNA is amplified in quantity before cloning, which process comprises:
(a) contacting each DNA with four different nucleoside triphosphates and
two oligonucleotide primers, for each different specific sequence being
amplified, wherein each primer is selected to be sufficiently
complementary to different strands of each specific sequence to hybridize
therewith, such that the extension product synthesized from one primer,
when separated from its complement, can serve as a template for synthesis
of the extension product of the other primer, and wherein each sequence
being amplified or each primer contains a restriction site, at a
temperature which promotes hybridization of each primer to its
complementary strand;
(b) contacting each strand, at the same time as or after step (a) or (b),
with a thermostable enzyme which catalyzes combination of the nucleoside
triphosphates to form primer extension products complementary to each
strand of DNA;
(c) maintaining the mixture from step (b) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand;
(d) heating the mixture from step (c) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized, to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(e) cooling the mixture from step (d) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced in step (d);
(f) maintaining the mixture from step (e) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand produced in step (d), but not
so high as to separate each extension product from its complementary
strand, steps (d), (e) and (f) being repeated a sufficient number of times
to result in detectable amplification of the DNA containing the
sequence(s), wherein steps (e) and (f) are carried simultaneously or
sequentially;
(g) adding to the product of step (f) a restriction enzyme for each of said
restriction sites to obtain cleaved products in a restriction digest; and
(h) ligating the cleaved product(s) of step (g) containing the specific
sequence(s) to be cloned into one or more cloning vectors.
37. A process for cloning into a vector at least one specific DNA sequence
contained in a DNA or a mixture of nucleic acids, wherein the DNA consists
of two complementary strands of equal or unequal length, and wherein the
DNA is amplified in quantity before cloning, which process comprises:
(a) heating the DNA in the presence of four different nucleoside
triphosphates and two oligonucleotide primers, for each different specific
sequence being amplified, for an effective time and at an effective
temperature to denature the DNA, wherein each primer is selected to be
sufficiently complementary to different strands of each specific sequence
to hybridize therewith, such that the extension product synthesized from
one primer, when separated from its complement, can serve as a template
for synthesis of the extension product of the other primer, and wherein
each sequence being amplified or each primer contains a restriction site;
cooling the denatured DNA to a temperature effective to promote
hybridization between each primer and its complementary strand;
(c) contacting the denatured DNA, at the same time as or after step (a) or
(b), with a thermostable enzyme which catalyzes combination of the
nucleoside triphosphates to form primer extension products complementary
to each strand of DNA;
(d) maintaining the mixture from step (c) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand;
(e) heating the mixture from step (d) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized, to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(f) cooling the mixture from step (e) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced in step (e);
(g) maintaining the mixture from step (f) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand produced in step (e), but not
so high as to separate each extension product from its complementary
strand, steps (e), (f), and (g) being repeated a sufficient number of
times to result in detectable amplification of the DNA containing the
sequences(s), wherein steps (f) and (g) are carried out simultaneously or
sequentially;
(h) adding to the product of step (g) a restriction enzyme for each of said
restriction sites to obtain cleaved products in a restriction digest; and
(i) ligating the cleaved product(s) of step (h) containing the specific
sequence(s) to be cloned into one or more cloning vectors containing a
selectable marker.
38. The process of clam 37, wherein steps (e)-(g) are repeated at least
five times.
39. The process of claim 37, further comprising the step of passing the
restriction digest of step (h) through a desalting column or membrane
before step (i).
40. The process of claim 37, further comprising, after step (i), sequencing
the cleaved product ligated into the vector.
41. The process of claim 37, wherein said specific DNA sequence encodes a
protein and wherein said method further comprises, after step (i),
expressing the protein encoded by the specific nucleic acid sequence.
42. The process of claim 37, wherein one specific sequence is being
amplified, the restriction sites are different on each primer, and the
product of step (h) is ligated into one cloning vector with a specific
orientation.
43. The process of claim 42, wherein the specific DNA sequence amplified is
or is contained within the .beta.-globin gene or the N-RAS oncogene.
44. The process of claim 37, wherein the thermostable enzyme is a
polymerase from Thermus aquaticus.
45. The process of claim 37, wherein the heating and cooling steps (e)-(g)
are automated by a machine which controls temperature levels, transitions
from one temperature to another, and the timing of the temperature levels.
46. The process of claim 36, wherein each primer contains a restriction
site which is the same as or different from the restriction site(s) on the
other primer(s).
47. The process of clam 46, wherein the restriction sites are on the 5' end
of the primers.
48. A process for cloning into a cloning vector one or more specific DNA
sequences contained in a DNA or mixture of nucleic acids, which DNA when
double-stranded consists of two separated complementary strands of equal
to unequal length and which DNA is amplified in quantity before cloning
which process comprises:
(a) contacting each DNA with four different nucleoside triphosphates and
two oligonucleotide primers, for each different specific sequence being
amplified, wherein each primer is selected to sufficiently complementary
to different strands of each specific sequence to hybridize therewith,
such that the extension product synthesized from one primer, when separatd
from its complement, can serev as a template for synthesis of teh
extension product of teh other primer, at a temperature which promotes
hybridization of each primer to its complementary strand;
(b) contacting each strand, at the same time as or after step (a) or (b),
with a thermostable enzyme which catalyzes combination of the nucleoside
triphosphates to form primer extension products complementary to each
strand of DNA;
(c) maintaining the mixture from step (b) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand;
(d) heating the mixture from step (c) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(e) cooling the mixture from step (d) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced in step (d);
(f) maintaining the mixture from step (e) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand produced in step (d), but not
so high as to separate each extension product from its complementary
strand, steps (d), (e) and (f) being repeated a sufficient number of times
to result in effective amplification of the DNA containing the sequence(s)
for blunt-end ligation into one or more cloning vectors, wherein steps (e)
and (f) are conducted simultaneously or sequentially; and
(g) ligating the amplified specific sequence(s) to be cloned obtained from
step (f) into one or more of said cloning vectors in the presence of a
ligase, said amplified sequence(s) and vector(s) being present in
sufficient amounts to effect the ligation.
49. A process for cloning into a cloning vector at least one specific DNA
sequence contained in a DNA or mixture of nucleic acids, which DNA
consists of two complementary strands of equal to unequal length and which
DNA is amplified in quantity before cloning, which process comprises:
(a) heating the DNA in the presence of four different nucleoside
triphophates and two oligonucleotide primers, for each different specific
sequencing being amplified, for an effective time and at an effective
temperature to denature the DNA, wherein each primer is selected be
sufficiently complementary to different strands of each specific sequence
to hybridize therewith, such that the extension product synthesized from
one primer, when separated from its complement, can serve as a template
for synthesis of the extension product of the other primer;
(b) cooling the denatured DNA to a temperature effective to promote
hybridization between each primer and its complementary strand;
(c) contacting the denatured DNA, at the same time as or after step (a) or
(b), with a thermostable enzyme which catalyzes combination of the
nucleoside triphosphates to form primer extension products complementary
to each strand of DNA;
(d) maintaining the mixture from step (c) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand, but not so high as to
separate each extension product from its complementary strand;
(e) heating the mixture from step (d) for an effective time and at an
effective temperature to separate the primer extension products from the
strands on which they were synthesized, to produce single-stranded
molecules, but not so high as to denature irreversibly the enzyme;
(f) cooling the mixture from step (e) for an effective time and to an
effective temperature to promote hybridization of each primer to its
complementary single-stranded molecule produced in step (e);
(g) maintaining the mixture from step (f) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each strand produced in step (e), but not
so high as to separate each extension product from its complementary
strand, steps (e), (f) and (g) being repeated a sufficient number of times
to result in effective amplification of the DNA containing each sequence
for bluntend ligation into one or more cloning vectors, wherein steps (f)
and (g) are conducted simultaneously or sequentially; and
(h) ligating the amplified specific sequence(s) to be cloned obtained from
step (g) into one or more of said cloning vectors in the presence of a
ligase, said amplified sequence(s) and vector(s) being present in
sufficient to effect the ligation.
50. The process of claim 49, wherein one DNA sequence is cloned into one
vector and two primers are employed. |
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Claims |
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Description |
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FIELD OF THE INVENTION
The present invention relates to a process for amplifying existing nucleic
acid sequences if they are present in a test sample and detecting them if
present by using a probe. More specifically, it relates to a process for
producing any particular nucleic acid sequence from a given sequence of
DNA or RNA in amounts which are large compared to the amount initially
present so as to facilitate detection of the sequences, using a
thermostable enzyme to catalyze the reaction. The DNA or RNA may be
single- or double-stranded, and may be a relatively pure species or a
component of a mixture of nucleic acids. The process of the invention
utilizes a repetitive reaction to accomplish the amplification of the
desired nucleic acid sequence.
DESCRIPTION OF RELATED DISCLOSURES
For diagnostic applications in particular, the target nucleic acid sequence
may be only a small portion of the DNA or RNA in question, so that it may
be difficult to detect its presence using nonisotopically labeled or
end-labeled oligonucleotide probes. Much effort is being expended in
increasing the sensitivity of the probe detection systems, but little
research has been conducted on amplifying the target sequence so that it
is present in quantities sufficient to be readily detectable using
currently available methods.
Several methods have been described in the literature for the synthesis of
nucleic acids de novo or from an existing sequence. These methods are
capable of producing large amounts of a given nucleic acid of completely
specified sequence.
One known method for synthesizing nucleic acids de novo involves the
organic synthesis of a nucleic acid from nucleoside derivatives. This
synthesis may be performed in solution or on a solid support. One type of
organic synthesis is the phosphotriester method, which has been utilized
to prepare gene fragments or short genes. In the phosphotriester method,
oligonucleotides are prepared which can then be joined together to form
longer nucleic acids. For a description of this method, see Narang, S.A.,
et al., Meth. Enzymol., 68, 90 (1979) and U.S. Pat. No. 4,356,270. The
patent describes the synthesis and cloning of the somatostatin gene.
A second type of organic synthesis is the phosphodiester method, which has
been utilized to prepare a tRNA gene. See Brown, E. L., et al., Meth.
Enzymol., 68, 109 (1979) for a description of this method. As in the
phosphotriester method, the phosphodiester method involves synthesis of
oligonucleotides which are subsequently joined together to form the
desired nucleic acid.
Although the above processes for de novo synthesis may be utilized to
synthesize long strands of nucleic acid, they are not very practical to
use for the synthesis of large amounts of a nucleic acid. Both processes
are laborious and time-consuming, require expensive equipment and
reagents, and have a low overall efficiency. The low overall efficiency
may be caused by the inefficiencies of the synthesis of the
oligonucleotides and of the joining reactions. In the synthesis of a long
nucleic acid, or even in the synthesis of a large amount of a shorter
nucleic acid, many oligonucleotides would need to be synthesized and many
joining reactions would be required. Consequently, these methods would not
be practical for synthesizing large amounts of any desired nucleic acid.
Methods also exist for producing nucleic acids in large amounts from small
amounts of the initial existing nucleic acid. These methods involve the
cloning of a nucleic acid in the appropriate host system, where the
desired nucleic acid is inserted into an appropriate vector which is used
to transform the host. When the host is cultured the vector is replicated,
and hence more copies of the desired nucleic acid are produced. For a
brief description of subcloning nucleic acid fragments, see Maniatis, T.,
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory., pp. 390401 (1982). See also the techniques described in U.S.
Pat. Nos. 4,416,988 and 4,403,036.
A third method for synthesizing nucleic acids, described in U.S. Pat. No.
4,293,652, is a hybrid of the above-described organic synthesis and
molecular cloning methods. In this process, the appropriate number of
oligonucleotides to make up the desired nucleic acid sequence is
organically synthesized and inserted sequentially into a vector which is
amplified by growth prior to each succeeding insertion.
The present invention bears some similarity to the molecular cloning
method; however, it does not involve the propagation of any organism and
thereby avoids the possible hazards or inconvenience which this entails.
The present invention also does not require synthesis of nucleic acid
sequences unrelated to the desired sequence, and thereby the present
invention obviates the need for extensive purification of the product from
a complicated biological mixture.
European Pat. Publication No. 200,362 published Dec. 10, 1986 discloses a
procedure whereby existing nucleic acids may be produced in larger
quantities so as to prepare other nucleic acids or to diagnose for the
presence of nucleic acids. The amplification and detection process is also
described by Saiki et al., Science, 230:1350-1354 (1985), and by Saiki et
al., Biotechnology, 3:1008-1012 (1985). Copending U.S. patent application
Ser. No. 899,061 filed Aug. 22, 1986, supra, discloses carrying out an
amplification of nucleic acids in the presence of a thermostable enzyme in
a heat-conducting block whose temperature is controlled by computer means.
Copending U.S. patent application Ser. No. 899,344 filed Aug. 22, 1986,
supra, discloses an amplification procedure followed by dot blot analysis
using a heatstable enzyme. Now abandoned U.S. application patent Ser. No.
899,241 filed Aug. 22, 1986, supra, discloses purification of a
thermostable enzyme, preferably a polymerase from Thermus aquaticus.
SUMMARY OF THE INVENTION
The present invention resides in a process for amplifying one or more
specific nucleic acid sequences present in a nucleic acid or mixture
thereof using primers and a thermostable enzyme. The extension product of
one primer when hybridized to the other becomes a template for the
production of the desired specific nucleic acid sequence, and vice versa,
and the process is repeated as often as is necessary to produce the
desired amount of the sequence. The method herein improves the specificity
of the amplification reaction, resulting in a very distinct signal of
amplified nucleic acid. In addition, the method herein eliminates the need
for transferring reagents from one vessel to another after each
amplification cycle. Such transferring is not required because the
thermostable enzyme will withstand the high temperatures required to
denature the nucleic acid strands and therefore does not need replacement.
The temperature cycling may, in addition, be automated for further
reduction in manpower and steps required to effectuate the amplification
reaction.
More specifically, the present invention provides a process for amplifying
at least one specific nucleic acid sequence contained in a nucleic acid or
a mixture of nucleic acids, wherein if the nucleic acid is
double-stranded, it consists of two separated complementary strands of
equal or unequal length, which process comprises:
(a) contacting each nucleic acid strand with four different nucleoside
triphosphates and one oligonucleotide primer for each different specific
sequence being amplified, wherein each primer is selected to be
substantially complementary to different strands of each specific
sequence, such that the extension product synthesized from one primer,
when it is separated from its complement, can serve as a template for
synthesis of the extension product of the other primer, said contacting
being at a temperature which promotes hybridization of each primer to its
complementary nucleic acid strand.,
(b) contacting each nucleic acid strand, at the same time as or after step
(a), with a thermostable enzyme which enables combination of the
nucleotide triphosphates to form primer extension products complementary
to each strand of each nucleic acid,
(c) maintaining the mixture from step (b) at an effective temperature for
an effective time to activate the enzyme, and to synthesize, for each
different sequence being amplified, an extension product of each primer
which is complementary to each nucleic acid strand template, but not so
high (a temperature) as to separate each extension product from its
complementary strand template;
(d) heating the mixture from step (c) for an effective time and at an
effective temperature to separate the primer extension products from the
templates on which they were synthesized to produce single-stranded
molecules, but not so high (a temperature) as to denature irreversibly the
enzyme;
(e) cooling the mixture from step (d) at an effective temperature for an
effective time to promote hybridization of each primer to each of the
single-stranded molecules produced in step (d); and
(f) maintaining the mixture from step (e) at an effective temperature for
an effective time to promote the activity of the enzyme and to synthesize,
for each different sequence being amplified, an extension product of each
primer which is complementary to each nucleic acid strand template
produced in step (d), but not so high (a temperature) as to separate each
extension product from its complementary strand template, wherein steps
(e) and (f) are carried out simultaneously or sequentially.
The steps (d), (e) and (f) may be repeated until the desired level of
sequence amplification is obtained. The preferred thermostable enzyme
herein is a polymerase extracted from Thermus aquaticus (Taq polymerase).
Most preferably, if the enzyme is Taq polymerase, in step (a) the nucleic
acid strands are contacted with a buffer comprising about 1.5-2 mM of a
magnesium salt, 150-200 .mu.M each of the nucleotides, and 1 nM of each
primer, steps (a), (e) and (f) are carried out at about 45-58.degree. C.,
and step (d) is carried out at about 90-100.degree. C.
In a preferred embodiment, the nucleic acid(s) are doublestranded and step
(a) is accomplished by (i) heating each nucleic acid in the presence of
four different nucleoside triphosphates and one oligonucleotide primer for
each different specific sequence being amplified, for an effective time
and at an effective temperature to denature each nucleic acid, wherein
each primer is selected to be substantially complementary to different
strands of each specific sequence, such that the extension product
synthesized from one pr | | |
