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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 nuclei 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. The DNA or RNA may be
single- or doublestranded, 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.
BACKGROUND OF THE INVENTION
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 nuclei 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. 390-401 (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.
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 agents for polymerization and then detecting the
amplified sequence. 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. This
method is expected to be more efficient than the methods described above
for producing large amounts of nucleic acid from a target sequence and to
produce such nucleic acid in a comparatively short period of time. The
present method is especially useful for amplifying rare species of nucleic
acid present in a mixture of nucleic acids for effective detection of such
species.
More specifically, the present invention provides a process for detecting
the presence or absence of at least one specific nucleic acid sequence in
a sample containing a nucleic acid or mixture of nucleic acids, or
distinguishing between two different forms of sequences in said sample,
wherein the sample is suspected of containing said sequence or sequences,
which process comprises:
(a) treating the sample with one oligonucleotide primer for each strand of
each different specific sequence suspected of being present in the sample,
under hybridizing conditions such that for each strand of each different
sequence to be detected an extension product of each primer is synthesized
which is complementary to each nucleic acid strand, wherein said primer or
primers are selected so as to be substantially complementary to each
strand 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;
(b) treating the sample under denaturing conditions to separate the primer
extension products from their templates if the sequence or sequences to be
detected are present;
(c) treating the sample with oligonucleotide primers such that a primer
extension product is synthesized using each of the single strands produced
in step (b) as a template, resulting in amplification of the specific
nucleic acid sequence or sequences if present;
(d) adding to the product of step (c) a labeled probe capable of
hybridizing to said sequence being detected or a mutation thereof; and
(e) determining whether said hybridization has occurred.
The steps (a)-(c) may be conducted sequentially or simultaneously. In
addition, steps (b) and (c) may be repeated until the desired level of
sequence amplification is obtained.
In other embodiments the invention relates to diagnostic kits for the
detection of at least one specific nucleic acid sequence in a sample
containing one or more nucleic acids at least one of which nucleic acid is
suspected of containing said sequence, which kit comprises, in packaged
form, a multicontainer unit having
(a) one container for each oligonucleotide primer for each strand of each
different sequence to be detected, which primer or primers are
substantially complementary to each strand of each specific nucleic acid
sequence such that an extension product synthesized from one primer, when
it is separated from its complement, can serve as a template for the
synthesis of the extension product of the other primer;
(b) a container containing an agent for polymerization;
(c) a container for each of four different nucleoside triphosphates;
(d) a container containing a probe capable of detecting the presence of
said sequence in said sample; and
(e) a container containing means for detecting hybrids of said probe and
said sequence.
In yet another embodiment, the invention relates to a process for cloning
into a vector a specific nucleic acid sequence contained in a nucleic acid
or a mixture of nucleic acids, which process comprises:
(a) treating the nucleic acid(s) with one oligonucleotide primer for each
strand of each different specific sequence being amplified, under
conditions such that for each strand of each different sequence being
amplified an extension product of each primer is synthesized which is
complementary to each nucleic acid strand, wherein said primer or primers
are selected so as to be substantially complementary to each strand of
each specific sequence such that the extension product synthesized form
one primer, when it is separated from its complement, can serve as a
template for synthesis of the extension product of the other primer, and
wherein said primer or primers each contain a restriction site on its 5'
end which is the same as or different from the restriction site(s) on the
other primer(s);
(b) separating the primer extension products from the templates on which
they are synthesized to produce single-stranded molecules;
(c) treating the single-stranded molecules generated from step (b) with
oligonucleotide primers such that a primer extension product is
synthesized using each of the single strands produced in step (b) as a
template, wherein depending on the particular sequence being amplified,
steps (a) and (c) are carried out in the presence of from 0 up to an
effective amount of dimethylsulfoxide or at a temperature of up to about
45.degree. C.;
(d) adding to the product of step (c) a restriction enzyme for each of said
restriction sites to obtain cleaved products in a restriction digest; and
(e) ligating the cleaved product(s) into one or more cloning vectors.
In yet another embodiment, the invention herein relates to a process for
synthesizing a nucleic acid fragment from an existing nucleic acid
fragment having fewer nucleotides than the fragment being synthesized and
two oligonucleotide primers, wherein the nucleic acid being synthesized is
comprised of a left segment, a core segment and a right segment, and
wherein the core segment represents at least substantially the nucleotide
sequence of said existing nucleic acid fragment, and the right and left
segments represent the sequence nucleotide present in the 5' ends of the
two primers, the 3' ends of which are complementary or substantially
complementary to the 3' ends of the single strands produced by separating
the strands of said existing nucleic acid fragment, which process
comprises:
(a) treating the strands of said existing fragment with two oligonucleotide
primers under conditions such that an extension product of each primer is
synthesized which is complementary to each nucleic acid strand, wherein
said primers are selected so as to be substantially complementary to the
3' end of each strand of said existing fragment 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, and wherein each primer contains, at its 5' end, a
sequence of nucleotides which are not complementary to said existing
fragment and which correspond to the two ends of the nucleic acid fragment
being synthesized;
(b) separating the primer extension products from the templates on which
they were synthesized to produce single-stranded molecules;
(c) treating the single-stranded molecules generated from step (b) with the
primers of step (a) under conditions such that a primer extension product
is synthesized using each of the single strands produced in step (b) as a
template so as to produce two intermediate double-stranded nucleic acid
molecules, into each of which has been incorporated the nucleotide
sequence present in the 5' end of one of the oligonucleotide primers, and
two full-length doublestranded nucleic acid molecules, into each of which
has been incorporated the nucleotide sequence present in the 5' ends of
both of the oligonucleotide primers;
(d) repeating steps (b) and (c) for a sufficient number of times to produce
the full-length double-stranded molecule in an effective amount;
(e) treating the strands of the product of step (d) with two primers so as
to lengthen the product of step (d) on both ends; and
(f) repeating steps (a)-(d) using the product of step (d) as the core
fragment and two oligonucleotide primers which are complementary or
substantially complementary to the 3' ends of the single strands produced
by separating the strands of the product of step (d).
The core fragment may be obtained by the steps comprising:
(a) reacting two oligonucleotides, each of which contain at their 3' ends a
nucleotide sequence which is complementary to the other oligonucleotide at
its 3' end, and which are non-complementary to each other at their 5'
ends, with an agent for polymerization and four nucleoside triphosphates
under conditions such that an extension product of each oligonucleotide is
synthesized which is complementary to each nucleic acid strand;
(b) separating the extension products from the templates on which they are
synthesized to produce single-stranded molecules; and
(c) treating the single-stranded molecules generated from step (b) with the
oligonucleotides of step (a) under conditions such that a primer extension
product is synthesized using each of the single strands produced in step
(b) as a template, resulting in amplification of the core fragment.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a 94 base pair length sequence of human .beta.-globin
desired to be amplified. The single base pair change which is associated
with sickle cell anemia is depicted beneath the 94-mer.
FIG. 2 illustrates a photograph of an ethidium bromidestained
polyacrylamide gel demonstrating amplification of the 94-mer contained in
human wild-type DNA and in a plasmid containing a 1.9 kb BamHI fragment of
the normal .beta.-globin gene (pBR328:HbA).
FIG. 3 illustrates a photograph of an ethidium bromidestained
polyacrylamide gel demonstrating amplification of any of the specific
target 94-mer sequence present in pBR328:HbA, a plasmid containing a 1.9
kb BamHI fragment of the sickel cell allele of .beta.-globin (pBR328:HbS),
pBR328:HbA where the sequence to be amplified is cleaved with MstII, and
pBR328:HbS where the sequence to be amplified has been treated but not
cleaved with MstII.
FIG. 4 illustrates in detail the steps and products of the polymerase chain
reaction for amplification of the desired 94-mer sequence of human
.beta.-globin for three cycles using two oligonucleotide primers.
FIG. 5 represents a photograph of an ethidium bromidestained polyacrylamide
gel demonstrating amplification after four cycles of a 240-mer sequence in
pBR328:HbA, where the aliquots are digested with NcoI (Lane 3), MstII
(Lane 4) or HinfI (Lane 5). Lane 1 is the molecular weight standard and
Lane 2 contains the intact 240-bp product.
FIG. 6 illustrates the sequence of the normal (.beta..sup.A) and sickle
cell (.beta..sup.S) .beta.-globin genes in the region of the DdeI and
HinfI restriction sites, where the single lines for .beta..sup.A mark the
position of the DdeI site (CTGAG) and the double bars for .beta..sup.A and
.beta..sup.S mark the position of the HinfI site (GACTC).
FIG. 7 illustrates the results of sequential digestion of normal
.beta.-globin using a 40-mer probe and DdeI followed by HinfI restriction
enzymes.
FIG. 8 illustrates the results of sequential digestion of sickel
.beta.-globin using the same 40-mer probe as in FIG. 7 and DdeI followed
by HinfI restriction enzymes.
FIG. 9 illustrates a photograph of an ethidium bromidestained
polyacrylamide gel demonstrating the use of the same 40-mer probe as in
FIG. 7 to specifically characterize the beta-globin alleles present in
samples of whole human DNA which have been subjected to amplification,
hybridization with the probe, and sequential digestion with DdeI and
HinFfI.
FIG. 10 illustrates a photograph of a 6% NuSieve agarose gel visualized
using ethidium bromide and UV light. This photograph demonstrates
amplfication of a sub-fragment of a 110-bp amplification product which
sub-fragment is an inner nested set within the 110-bp fragment.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The term "oligonucleotide" as used herein in referring to primers, probes,
oligomer fragments to be detected, oligomer controls and unlabeled
blocking oligomers is defined as a molecule comprised of two or more
deoxyribonucleotides or ribonucleotides, preferably more than three. Its
exact size will depend on many factors, which in turn depend on the
ultimate function or use of the oligonucleotide.
The term "primer" as used herein refers to an oligonucleotide whether
occurring naturally as in a purified restriction digest or produced
synthetically, which is capable of acting as a point of initiation of
synthesis when placed under conditions in which synthesis of a primer
extension product which is complementary to a nucleic acid strand is
induced, i.e., in the presence of nucleotides and an agent for
polymerization such as DNA polymerase and at a suitable temperature and
pH. The primer is preferably single stranded for maximum efficiency in
amplification, but may alternatively be double stranded. If double
stranded, the primer is first treated to separate its strands before being
used to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to prime
the synthesis of extension products in the presence of the agent for
polymerization. The exact lengths of the primers will depend on many
factors, including temperature and source of primers. For example,
depending on the complexity of the target sequence, the oligonucleotide
primer typically contains 15-25 or more nucleotides, although it may
contain fewer nucleotides. Short primer molecules generally require cooler
temperatures to form sufficiently stable hybrid complexes with template.
The primers herein are selected to be "substantially" complementary to the
different strands of each specific sequence to be amplified. This means
that the primers must be sufficiently complementary to hybridize with
their respective strands. Therefore, the primer sequence need not reflect
the exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the strand.
Alternatively, noncomplementary bases or longer sequences can be
interspersed into the primer, provided that the primer sequence has
sufficient complementarity with the sequence of the strand to be amplified
to hybridize therewith and thereby form a template for synthesis of the
extension product of the other primer.
As used herein, the terms "restriction endonucleases" and "restriction
enzymes" refer to bacterial enzymes each of which cut double-stranded DNA
at or near a specific nucleotide sequence.
As used herein, the term "DNA polymorphism" refers to the condition in
which two or more different nucleotide sequences coexist in the same
interbreeding population in a DNA sequence.
The term "restriction fragment length polymorphism" ("RFLP") refers to the
differences in DNA nucleotide sequences that are randomly distributed
throughout the entire human genome and that produce different restriction
endonuclease patterns.
The present invention is directed to a process for amplifying any one of
more desired specific nucleic acid sequences suspected of being in a
nucleic acid. Because large amounts of a specific sequence may be produced
by this process, the present invention may be used for improving the
efficiency of cloning DNA or messenger RNA and for amplifying a target
sequence to facilitate detection thereof.
In general, the present process involves a chain reaction for producing, in
exponential quantities relative to the number of reaction steps involved,
at least one specific nucleic acid sequence given (a) that the ends of the
required sequence are known in sufficient detail that oligonucleotides can
be synthesized which will hybridize to them, and (b) that a small amount
of the sequence is available to initiate the chain reaction. The product
of the chain reaction will be a discrete nucleic acid duplex with termini
corresponding to the ends of the specific primers employed.
Any source of nucleic acid, in purified or nonpurified form, can be
utilized as the starting nucleic acid or acids, provided it is suspected
of containing the specific nucleic acid sequence desired. Thus, the
process may employ, for example, DNA or RNA, including messenger RNA,
which DNA or RNA may be single stranded or double stranded. In addition, a
DNA-RNA hybrid which contains one strand of each may be utilized. A
mixture of any of these nucleic acids may also be employed, or the nucleic
acids produced from a previous amplification reaction herein using the
same or different primers may be so utilized. The specific nucleic acid
sequence to be amlified may be only a fraction of a larger molecule or can
be present initially as a discrete molecule, so that the specific sequence
constitutes the entire nucleic acid. It is not necessary that the sequence
to be amplified be present initially in a pure form; it may be a minor
fraction of a complex mixture, such as a portion of the .beta.-globin gene
contained in whole human DNA or a portion of nucleic acic sequence due to
a particular microorganism which organism might constitute only a very
minor fraction of a particular biological sample. The starting nucleic
acid may contain more than one desired specific nucleic acid sequence
which may be the same or different. Therefore, the present process is
useful not only for producing large amounts of one specific nucleic acid
sequence, but also for amplifying simultaneously more than one different
specific nucleic acid sequence located on the same or different nucleic
acid molecules.
The nucleic acid or acids may be obtained from any source, for example,
from plasmids such as pBR322, from cloned DNA or RNA, or from natural DNA
or RNA from any source, including bacteria, yeast, viruses, and higher
organisms such as plants or animals. DNA or RNA may be extracted from
blood, tissue material such as chorionic villi or amniotic cells by a
variety of techniques such as that described by Maniatis et al., Molecular
Cloning: A Laboratory Manual, (New York: Cold Spring Harbor Laboratory,
1982), pp. 280-281.
Any specific nucleic acid sequence can be produced by the present process.
It is only necessary that a sufficient number of bases at both ends of the
sequence be known in sufficient detail so that two oligonucleotide primers
can be prepared which will hybridize to different strands of the desired
sequence and at relative positions along the sequence such that an
extension product synthesized from one primer, when it is separated from
its template (complement), can serve as a template for extension of the
other primer into a nucleic acid of defined length. The greater the
knowledge about the bases at both ends of the sequence, the greater can be
the specificity of the primers for the target nucleic acid sequence, and
thus the greater the efficiency of the process. It will be understood that
the word primer as used hereinafter may refer to more than one primer,
particularly in the case where there is some ambiguity in the information
regarding the terminal sequence(s) of the fragment to be amplified. For
instance, in the case where a nucleic acid sequence is inferred from
protein sequence information a collection of primers containing sequences
representing all possible codon variations based on degeneracy of the
genetic code will be used for each strand. One primer from this collection
will be homologous with the end of the desired sequence to be amplified.
The oligonucleotide primers may be prepared using any suitable method, such
as, for example, the phosphotriester and phosphodiester methods described
above, or automated embodiments thereof. In one such automated embodiment
diethylphosphoramidites are used as starting materials and may be
synthesized as described by Beaucage et al., Tetrahedron Letters (1981),
22:1859-1862. One method for synthesizing oligonucleotides on a modified
solid support is described in U.S. Pat. No. 4,458,066. It is also possible
to use a primer which has been isolated from a biological source (such as
a restriction endonuclease digest).
The specific nuclei acid sequence is produced by using the nucleic acid
containing that sequence as a template. If the nucleic acid contains two
strands, it is necessary to separate the strands of the nucleic acid
before it can be used as the template, either as a separate step or
simultaneously with the synthesis of the primer extension products. This
strand separation can be accomplished by any suitable denaturing method
including physical, chemical or enzymatic means. One physical method of
separating the strands of the nucleic acid involves heating the nucleic
acid until it is completely (>99%) denatured. Typical heat denaturation
may involve temperatures ranging from about 80.degree. to 105.degree. C.
for times ranging from about 1 to 10 minutes. Strand separation may also
be induced by an enzyme from the class of enzymes known as helicases or
the enzyme ReCA, which has helicase activity and in the presence of
riboATP is known to denature DNA. The reaction conditions suitable for
separating the strands of nucleic acids with helicases are described by
Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLIII "DNA:
Replication and Recombination" (New York: Cold Spring Harbor Laboratory,
1978), B. Kuhn et al., "DNA Helicases", pp. 63-67, and techniques for
using RecA are reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37
(1982).
If the original nucleic acid containing the sequence to be amplified is
single stranded, its complement is synthesized by adding one or two
oligonucleotide primers thereto. If an appropriate single primer is added,
a primer extension product is synthesized in the presence of the primer,
an agent for polymerization and the four nucleotides described below. The
product will be partially complementary to the single-stranded nucleic
acid and will hybridize with the nucleic acid strand to form a duplex of
unequal length strands that may then be separated into single strands as
described above to produce two single separated complementary strands.
Alternatively, two appropriate primers may be added to the single-stranded
nucleic acid and the reaction carried out.
If the original nucleic acid constitutes the sequence to be amplified, the
primer extension product(s) produced will be completely complementary to
the strands of the original nucleic acid and will hybridize therewith to
form a duplex of equal length strands to be separated into single-stranded
molecules.
When the complementary strands of the nucleic acid or acids are separated,
whether the nucleic acid was originally double or single stranded, the
strands are ready to be used as a template for the synthesis of additional
nucleic acid strands. This synthesis can be performed using any suitable
method. Generally it occurs in a buffered aqueous solution, preferably at
a pH of 7-9, most preferably about 8. Preferably, a molar excess (for
cloned nucleic acid, usually about 1000:1 primer:template, and for genomic
nucleic acid, usually about 10.sup.6 :1 primer:template) of the two
oligonucleotide primers is added to the buffer containing the separated
template strands. It is understood, however, that the amount of
complementary strand may not be known if the process herein is used for
diagnostic applications, so that the amount of primer relative to the
amount of complementary strand cannot be determined with certainty. As a
practical matter, however, the amount of primer added will generally be in
molar excess over the amount of complementary strand (template) when the
sequence to be amplified is contained in a mixture of complicated
long-chain nucleic acid strands. A large molar excess is preferred to
improve the efficiency of the process.
The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also
added to the synthesis mixture in adequate amounts and the resulting
solution is heated to about 90.degree.-100.degree. C. for from about 1 to
10 minutes, preferably from 1 to 4 minutes. After this heating period the
solution is allowed to cool to from 20.degree.-40.degree. C., which is
preferable for the primer hybridization. To the cooled mixture is added an
agent for polymerization, and the reaction is allowed to occur under
conditions known in the art. This synthesis reaction may occur at from
room temperature up to a temperature above which the agent for
polymerization no longer functions efficiently. Thus, for example, if DNA
polymerase is used as the agent for polymerization, the temperature is
generally no greater than about 45.degree. C. Preferably an amount of
dimethylsulfoxide (DMSO) is present which is effective in detection of the
signal or the temperature is 35.degree.-40.degree. C. Most preferably
5-10% by volume DMSO is present and the temperature is
35.degree.-40.degree. C. For certain applications, where the sequences to
be amplified are over 110 base pair fragments, such as the HLA DQ-.alpha.
or -.beta. genes, an effective amount (e.g., 10% by volume) of DMSO is
added to the amplification mixture, and the reaction is carried at
35.degree.-40.degree. C., to obtain detectable results or to enable
cloning.
The agent for polymerization may be any compound or system which will
function to accomplish the synthesis of primer extension products,
including enzymes. Suitable enzymes for this purpose include, for example,
E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4
DNA polymerase, other available DNA polymerases, reverse transcriptase,
and other enzymes, including heatstable enzymes, which will facilitate
combination of the nucleotides in the proper manner to form the primer
extension products which are complementary to each nucleic acid strand.
Generally, the synthesis will be initiated at the 3' end of each primer
and proceed in the 5' direction along the template strand, until synthesis
terminates, producing molecules of different lengths. There may be agents,
however, which initiate synthesis at the 5' end and proceed in the other
direction, using the same process as described above.
The newly synthesized strand and its complementary nucleic acid strand form
a double-stranded molecule which is used in the succeeding steps of the
process. In the next step, the strands of the double-stranded molecule are
separated using any of the procedures described above to provide
single-stranded molecules.
New nucleic acid is synthesized on the single-stranded molecules.
Additional inducing agent, nucleotides and primers may be added if
necessary for the reaction to proceed under the conditions prescribed
above. Again, the synthesis will be initiated at one end of the
oligonucleotide primers and will proceed along the single strands of the
template to produce additional nucleic acid. After this step, half of the
extension product will consist of the specific nucleic acid sequence
bounded by the two primers.
The steps of strand separation and extension product synthesis can be
repeated as often as needed to produce the desired quantity of the
specific nucleic acid sequence. As will be described in further detail
below, the amount of the specific nucleic acid sequence produced will
accumulate in an exponential fashion.
When it is desired to produce more than one specific nucleic acid sequence
from the first nucleic acid or mixture of nucleic acids, the appropriate
number of different oligonucleotide primers are utilized. For example, if
two different specific nucleic acid sequences are to be produced, four
primers are utilized. Two of the primers are specific for one of the
specific nucleic acid sequences and the other two primers are specific for
the second specific nucleic acid sequence. In this manner, each of the two
different specific sequences can be produced exponentially by the present
process.
The present invention can be performed in a step-wise fashion where after
each step new reagents are added, or simultaneously, where all reagents
are added at the initial step, or partially step-wise and partially
simultaneous, where fresh reagent is added after a given number of steps.
If a method of strand separation, such as heat, is employed which will
inactivate the agent for polymerization, as in the case of a heat-labile
enzyme, then it is necessary to replenish the agent for polymerization
after every strand separation step. The simultaneous method may be
utilized when a number of purified components, including an enzymatic
means such as helicase, is used for the strand separation step. In the
simultaneous procedure, the reaction mixture may contain, in addition to
the nucleic acid strand(s) containing the desired sequence, the
strandseparating enzyme (e.g., helicase), an appropriate energy source for
the strand-separating enzyme, such as rATP, the four nucleotides, the
oligonucleotide primers in molar excess, and the inducing agent, e.g.,
Klenow fragment of E. coli DNA polymerase I. If heat is used for
denaturation in a simultaneous process, a heat-stable inducing agent such
as a thermostable polymerase may be employed which will operate at an
elevated temperature, preferably 65.degree.-90.degree. C. depending on the
inducing agent, at which temperature the nucleic acid will consist of
single and double strands in equilibrium. For smaller lengths of nucleic
acid, lower temperatures of about 50.degree. C. may be employed. The upper
temperature will depend on the temperature at which the enzyme will
degrade or the temperature above which an insufficient level of primer
hybridization will occur. Such a heat-stable enzyme is described, e.g., by
A. S. Kaledin et al., Biokhimiya, 45, 644-651 (1980). Each step of the
process will occur sequentially notwithstanding the initial presence of
all the reagents. Additional materials may be added as necessary. After
the appropriate length of time has passed to produce the desired amount of
the specific nucleic acid sequence, the reaction may be halted by
inactivating the enzymes in any known manner or separating the components
of the reaction.
The process of the present invention may be conducted continuously. In one
embodiment of an automated process, the reaction may be cycled through a
denaturing region, a reagent addition region, and a reaction region. In
another embodiment, the enzyme used for the synthesis of primer extension
products can be immobilized in a column. The other reaction components can
be continuously circulated by a pump through the column and a heating coil
in series; thus the nucleic acids produced can be repeatedly denatured
without inactivating the enzyme.
The present invention is demonstrated diagrammatically below where
double-stranded DNA containing the desired sequence [S] comprised of
complementary strands [S.sup.+ ] and [S.sup.- ] is utilzied as the nucleic
acid. During the first and each subsequent reaction cycle extension of
each oligonucleotide primer on the original template will produce one new
ssDNA molecule product of indefinite length which terminates with only one
of the primers. These products, hereafter referred to as "long products,"
will accumulate in a linear fashion; taht is, the amount present after any
number of cycles will be proportional to the number of cycles.
The long products thus produced will act as templates for one or the other
of the oligonucleotide primers during subsequent cycles and will produce
molecules of the desired sequence [S.sup.+ ] or [S.sup.- ]. These
molecules will also function as templates for one of the other of the
oligonucleotide primers, producing further [S.sup.+ ] and [S.sup.- ], and
accumulation of [S] at an exponential rate relative to the number of
cycles.
By-products formed by oligonucleotide hybridizations other than those
intended are not self-catalytic (except in rare instances) and thus
accumulates at a linear rate.
The specific sequence to be amplified, [S], can be depicted
diagrammatically as:
##STR1##
It is seen that each strand which terminates with the oligonucleotide
sequence of one primer and the complementary sequence of the other is the
specific nucleic acid sequence [S] that is desired to be produced.
The steps of this process can be repeated indefinitely, being limited only
by the amount of Primers 1 and 2, the agent for polymerization and
nucleotides present. For detection, the number of cycles used is that
required to produce a detectable signal, an amount which will depend,
e.g., on the nature of the sample. For example, if the sample is pure or
diluted, fewer cycles may be required than if it is a complex mixture. If
the sample is human genomic DNA, preferably the number of cycles is from
about 10-30.
The amount of original nucleic acid remains constant in the entire process,
because it is not replicated. The amount of the long products increases
linearly because they are produced only from the original nucleic acid.
The amount of the specific sequence increases exponentially. Thus, the
specific sequence will become the predominant species. This is illustrated
in the following table, which indicates the relative amounts of the
species theor | | |
