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Description  |
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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a process for amplifying existing nucleic
acid sequences. 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. 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.
2. 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. 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 inducing agents. 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 amplifying
at least one specific nucleic acid sequence contained in a nucleic acid or
a mixture of nucleic acids wherein each nucleic acid consists of two
separate complementary strands, of equal or unequal length, which process
comprises:
(a) treating the strands with two primers, for each different specific
sequence being amplified, under conditions such that for each different
sequence being amplified 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
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;
(b) separating the primer extension products from the templates on which
they were synthesized to produce single-stranded molecules; and
(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.
The steps 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 methods for diagnosing the
presence of specific nucleic acid sequences suspected of being in a sample
and diagnostic kits applicable thereto.
The present invention may be useful not only for producing large amounts of
an existing nucleic acid of completely specified sequence, but also for
producing nucleic acid sequences which are known to exist but are not
completely specified. In either case an initial copy of the sequence to be
amplified must be available, although it need not be pure or a discrete
molecule.
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 an autoradiograph of polyacrylamide gel electrophoresis
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 an autoradiograph of polyacrylamide gel electrophoresis
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
sickle 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 the amplified has been treated but not cleaved with MstII.
FIGS. 4-1-4-3 illustrate 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 an autoradiograph of polyacrylamide gel electrophoresis
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 sickle
.beta.-globin using the same 40-mer probe as in FIG. 7 and DdeI followed
by HinfI restriction enzymes.
FIG. 9 illustrates an autoradiograph of polyacrylamide gel electrophoresis
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 by the present
method.
FIG. 10 illustrates a photograph of a 6% NuSieve agarose gel visualized
using ethidium bromide and UV light. This photograph demonstrates
amplification 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 inducing agent 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
firt 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 inducing agent. The exact lengths of the
primers will depend on many factors, including temperature, source of
primer and use of the method. For example, for diagnostics applications,
depending on the complexity of the target sequence, the oligonucleotide
primer typically contains 15-25 or more nucleotides, although it may
contain fewer nucleotides. For other applications, the oligonucleotide
primer is typically shorter, e.g., 7-15 nucleotides. Such 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, non-complementary 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 coexists 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 or
more desired specific nucleic acid sequences found 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. The present invention is also useful for
obtaining large amounts of the desired sequence from a mixture of nucleic
acids resulting from an imperfect chemical synthesis.
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 contains or 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
acid produced from a previous amplification reaction herein using the same
or different primers may be so utilized. The specific nucleic acid
sequence to be amplified 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
acid 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 100% 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: 1859-1962. 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 nucleic 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 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 inducer or catalyst of the synthesis 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 room temperature, which is preferable for
the primer hybridization. To the cooled mixture is added an appropriate
agent for inducing or catalyzing the primer extension reaction, 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 inducing agent no longer functions efficiently. Thus, for
example, if DNA polymerase is used as inducing agent, the temperature is
generally no greater than about 40.degree. C. Most conveniently the
reaction occurs at room temperature.
The inducing agent 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 heat-stable 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
inducing 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 inducing agent, as in the case of a heat-labile enzyme,
then it is necessary to replenish the inducing agent after every strand
separation step. The simultaneous method may be utilized when an enzymatic
means 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 strand-separating
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 were
double-stranded DNA containing the desired sequence [S] comprised of
complementary strands [S.sup.+ ] and [S.sup.- ] is utilized 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; that 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 or the other of the
oligonucleotide primers, producing further [S.sup.+ ] and [S.sup.- ], and
thus a chain reaction can be sustained which will result in the
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
accumulate at a linear rate.
The specific sequence to be amplified, [S], can be depicted
diagrammatically as:
__________________________________________________________________________
[S.sup.+ ] 5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3'
[S.sup.- ] 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
The appropriate oligonucleotide primers would be:
Primer 1: GGGGGGGGGG
Primer 2: AAAAAAAAAA
so that if DNA containing [S]
. . . zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz . .
. . . zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz . .
__________________________________________________________________________
is separated into single strands and its single strands are hybridized to
Primers 1 and 2, the following extension reactions can be catalyzed by DNA
polymerase in the presence of the four deoxyribonucleoside triphosphates:
##STR1##
On denaturation of the two duplexes formed, the products are:
##STR2##
If these four strands are allowed to rehybridize with Primers 1 and 2 in
the next cycle, inducing agent will catalyze the following reactions:
##STR3##
If the strands of the above four duplexes are separated, the following
strands are found:
__________________________________________________________________________
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3'
newly synthesized [S.sup.+ ]
3' . . . zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
first cycle synthesized long product 1
3' . . . zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
newly synthesized long product 1
5' . . . zzzzzzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzz . .
. 3'
original template strand.sup.+
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz . . . 3'
newly synthesized long product 2
3' . . . zzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz .
. . 5'
original template strand.sup.-
3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5'
newly synthesized [S.sup.- ]
5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzz . . . 3'
first cycle synthesized long product 2
__________________________________________________________________________
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, inducing agent and nucleotides present.
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 theoretically present after n cycles, assuming 100%
efficiency at each cycle:
______________________________________
Number of Double Strands
After 0 to n Cycles
Long Specific
Cycle Number Template Products Sequence [S]
______________________________________
0 1 -- --
1 1 1 0
2 1 2 1
3 1 3 4
5 1 5 26
10 1 10 1013
15 1 15 32,752
20 1 20 1,048,555
n 1 n (2.sup.n -n-1)
______________________________________
When a single-stranded nucleic acid is utilized as the template, only one
long product is formed per cycle.
The method herein may be utilized to clone a particular nucleic acid
sequence for insertion into a suitable expression vector. The vector may
then be used to transform an appropriate host organism to produce the gene
product of the sequence by standard methods of recombinant DNA technology.
In addition, the process herein can be used for in vitro mutagenesis. The
oligodeoxyribonucleotide primers need not be exactly complementary to the
DNA sequence which is being amplified. It is only necessary that they be
able to hydridize to the sequence sufficiently well to be extended by the
polymerase enzyme or by whatever other inducing agent is employed. The
product of a polymerase chain reaction wherein the primers employed are
not exactly complementary to the original template will contain the
sequence of the primer rather than the template, thereby introducing an in
vitro mutation. In further cycles this mutation will be amplified with an
undiminished efficiency because no further mispaired primings are
required. The mutant thus produced may be inserted into an appropriate
vector by standard molecular biological techniques and might confer mutant
properties on this vector such as the potential for production of an
altered protein.
The process of making an altered DNA sequence as described above could be
repeated on the altered DNA using different primers so as to induce
further sequence changes. In this way a series of mutated sequences could
gradually be produced wherein each new addition to the series could differ
from the last in a minor way, but from the original DNA source sequence in
an increasingly major way. In this manner changes could be made ultimately
which were not feasible in a single step due to the inability of a very
seriously mismatched primer to function.
In addition, the primer can contain as part of its sequence a
non-complementary sequence provided that a sufficient amount of the primer
contains a sequence which is complementary to the strand to be amplified.
For example, a nucleotide sequence which is not complementary to the
template sequence (such as, e.g., a promoter, linker, coding sequence,
etc.) may be attached at the 5' end of one or both of the primers, and
thereby appended to the product of the amplification process. After the
extension primer is added, sufficient cycles are run to achieve the
desired amount of new template containing the non-complementary nucleotide
insert. This allows production of large quantities of the combined
fragments in a relatively short period of time (e.g., two hours or less)
using a simple technique.
The method herein may also be used to enable detection and/or
characterization of specific nucleic acid sequences associated with
infectious diseases, genetic disorders or cellular disorders such as
cancer. Amplification is useful when the amount of nucleic acid available
for analysis is very small, as, for example, in the prenatal diagnosis of
sickle cell anemia using DNA obtained from fetal cells. Amplification is
particularly useful if such an analysis is to be done on a small sample
using non-radioactive detection techniques which may be inherently
insensitive, or where radioactive techniques are being employed but where
rapid detection is desirable.
For purposes of this invention genetic diseases may include specific
deletions and/or mutations in genomic DNA from any organism, such as,
e.g., sickle cell anemia, cystic fibrosis, .alpha.-thalassemia,
.beta.-thalassemia, and the like. Sickle cell anemia can be readily
detected via oligomer restriction analysis or a RFLP-like analysis
following amplification of the appropriate DNA sequence by the present
method. .alpha.-Thalassemia can be detected by the absence of a sequence,
and .beta.-thalassemia can be detected by the presence of a polymorphic
restriction site closely linked to a mutation which causes the disease.
All of these genetic diseases may be detected by amplifying the appropriate
sequence and analyzing it by Southern blots without using radioactive
probes. In such a process, for example, a small sample of DNA from, e.g.,
amniotic fluid containing a very low level of the desired sequence is
amplified, cut with a restriction enzyme, and analyzed via a Southern
blotting technique. The use of non-radioactive probes is facilitated by
the high level of the amplified signal.
In another embodiment a small sample of DNA may be amplified to a
convenient level and then a further cycle of extension reactions performed
wherein nucleotide derivatives which are readily detectable (such as
.sup.32 P-labeled or biotin labeled nucleoside triphosphates) are
incorporated directly into the final DNA product, which may be analyzed by
restriction and electrophoretic separation or any other appropriate
method. An example of this technique in a model system is demonstrated in
FIG. 5.
In a further embodiment, demonstrated in a model system in FIG. 3, the
nucleic acid may be exposed to a particular restriction endonuclease prior
to amplification. Since a sequence which has been cut cannot be amplified,
the appearance of an amplified fragment, despite prior restriction of the
DNA sample, implies the absence of a site for the endonuclease within the
amplified sequence. The presence or absence of an amplified sequence can
be detected by an appropriate method.
A practical application of this technique can be illustrated by its use in
facilitating the detection of sickle cell anemia via the oligomer
restriction technique described herein and in copending U.S. application
Ser. No. 716,982 to Erlich et al. entitled "Method For Detection of
Polymorphic Restriction Sites and Nucleic Acid Sequences" filed Mar. 28,
1985. Sickle cell anemia is a hemoglobin disease which is caused by a
single base pair change in the sixth codon of the .beta.-gl | | |
