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BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods and compositions for detecting the
presence or absence of a target DNA sequence, such as a mutation, within
an identified region of a selected DNA molecule, such as a gene. In
particular aspects, the invention relates to the use of novel primer
constructs in connection with the polymerase chain reaction (PCR)
technique for the detection of genetic mutations in genes, particularly
point mutations.
2. Description of the Related Art
The ability to detect specific nucleotide alterations or mutations in DNA
sequences such as genes is an invaluable tool for medical science. The
ability to identify such alterations provides a means for diagnosis of
genetic diseases that involve DNA mutations, including sickle-cell anemia,
thalassemia, diabetes, certain oncogenic mutations, and the like.
Importantly, the ability to diagnose genetic diseases such as the
foregoing would provide numerous advantages, ranging from the ability to
prepare for proper care and treatment of affected individuals, such as in
the case of prenatal diagnosis, to marital counseling of prospective
parents. Unfortunately, the techniques presently available to medical
science for such diagnosis have been generally quite limited in one or
more aspects.
One technique which has been used with some frequency employs the use of
the PCR or site-specific DNA amplification technique, in combination with
synthetic oligodeoxynucleotides. This technique, exemplified by the
procedure set forth in Verlaan-de-Vries, et al.: A dot blot screening
procedure for mutated ras oncogenes using synthetic oligonucleotides (Gene
50:313-320, 1986), involves the specific in vitro amplification of genetic
regions suspected of containing a particular, known mutation in a specific
configuration, followed by hybridization of the amplified DNA under
tightly controlled parameters with one or more oligonucleotides which
carry complementary mutations. By determining which of the
oligonucleotides bind tightly under the specified hybridization
conditions, one can attempt to ascertain which, if any, of the mutations
are present in the segment of the DNA that is amplified. While this
technique has shown some usefulness, it is quite cumbersome in that it
requires several steps, including both an amplification step followed by a
separate hybridization step. Furthermore, the technique relies upon very
tightly controlled hybridization conditions, thus rendering it generally
inapplicable to everyday clinical application.
A second approach which has found some usefulness in connection with
certain genetic disorders involves the use of restriction enzyme analysis
of DNA to identify changes in restriction fragmentation pattern of the
suspected or selected DNA in comparison with a standard or reference DNA.
In one approach employing restriction enzyme analysis, the selected and
reference DNAs are simply compared, side-by-side, using various
restriction enzyme digestions. An alteration in the digestion pattern of
the selected DNA versus the reference DNA is indicative of a mutation,
such as an insertion or deletion.
However, to identify very small mutations, such as point mutations or
insertions or deletions of very short regions, the foregoing restriction
enzyme analysis approach is often inapplicable. This is because it is very
difficult to identify very small shifts in molecular weight of DNA
fragments. In fact, in the case of very small mutations, restriction
enzyme analysis is typically only applicable where the mutation involves
the restriction enzyme target site itself. While some examples are known
where direct restriction analysis is useful for identifying mutations, the
foregoing technique is generally not applicable to a broad range of
embodiments and is therefore quite limited in its usefulness. For example,
while in the case of sickle-cell anemia the precise mutation is generally
known (Saiki, et al.: Enzymatic amplification of beta-globin genomic
sequences and restriction site analysis for diagnosis of sickle cell
anemia. Science 230(1732):1350-4, 1985), in B-thalassemia, over 40
different possible mutations are known, generally ranging from about 1 to
3 basepairs. Therefore, this technique is not applicable where the genetic
disease fails to create either a changed or new restriction site or where
restriction fragment length polymorphism is not present.
An example of the foregoing direct restriction endonuclease analysis
approach is disclosed in U.S. Pat. No. 4,395,486 to Wilson et al. The
Wilson et al. patent teaches a method for the diagnosis of sickle cell
anemia by a restriction endonuclease assay through the use of an enzyme,
such as Dde I, which recognizes nucleotide base sequence CTNAG. The Wilson
et al. method relies upon the discovery that the sickle cell trait
involves a point mutation of the DNA at codon 6 of the Beta globin gene.
In normal individuals, codons 5 and 6 comprise the nucleotides CCT GAG,
respectively. However, in the affected individuals, there is an A to T
point mutation in codon 6 resulting in the changed sequence, CCT GTG.
Thus, enzymes such as Dde recognize and cleave DNA from the unaffected
individual, but not DNA from the affected individual.
A third, and quite complicated technique, has recently been described by
Landegren, et al.: A ligase-mediated gene detection technique. (Science 24
: 1077-1080, 1989). This technique is quite complicated because it
involves the dual hybridization of both a probing sequence, bearing a tag
such as an antibody tag, in combination with a second, labeled probing
sequence. This technique relies upon the ability to hybridize two
fragments in an adjacent position along a selected region of a DNA strand,
such as a gene suspected of containing a particular mutation. The mutation
is detected by the ability to ligate the probe bearing the mutation and
tag to the separate probe bearing the label. Ligation will be achieved
only where the mutation-bearing probe aligns in a position adjacent to the
label-bearing probe. As one might imagine, such a technique could only be
performed with great difficulty, involving many steps and is likely not
practical in the case of a clinical laboratory application.
The present invention involves a dramatic improvement over the foregoing
approaches to DNA analysis and relies in part on the PCR technique. The
PCR technique, described in U.S. Pat. Nos. 4,683,202 and 4,683,195,
involves a process for amplifying any desired specific nucleic acid
sequence contained in a nucleic acid sequence within a nucleic acid
molecule. The process includes treating separate complementary strands of
the nucleic acid with an excess of two oligonucleotide primers, and
extending the primers to form complementary primer extension products
which subsequently act as templates for synthesizing the desired nucleic
acid sequence. The steps of the PCR reaction may be carried out stepwise
and simultaneously, and can be repeated as often as desired in order to
achieve increased levels of amplification of the desired sequence. By this
technique, the sequence of DNA between the primers on the respective DNA
strands will be amplified selectively over the remaining portions of the
DNA in the selected sample. The PCR technique therefore provides for
specific amplification of a desired region of the DNA. Unfortunately,
while the PCR technique itself can amplify desired regions, it cannot
directly identify the nature of the sequences contained within such a
region.
Accordingly, it is apparent that there is needed a new approach that is
applicable to identifying DNA mutations, particular short mutations such
as point mutations or insertions or deletions of short regions. In
particular, there is needed a technique which has broad applicability to a
wide variety of mutations, such as to point and other short mutations,
which can be performed by a technician with minimal training and with
minimal difficulty and complexity of manipulation. The present invention
addresses these inadequacies in the art by providing such a technique
which can generally be applied to any mutation having a known position or
configuration and which can be performed simply with relatively few steps.
SUMMARY OF THE INVENTIONS
The present invention addresses these and/or other inadequacies in the
prior art by providing a technique for detecting the presence or absence
of a target DNA sequence within an identified region of a selected DNA
molecule. The technique of the present invention relies in part on the PCR
in vitro amplification procedure to amplify preferentially regions which
contain the target sequence or, alternatively or in addition, to amplify
selectively regions which do not contain the target sequence. In a general
and overall sense, the key to the present invention is the use of a
nucleic acid primer molecule which is capable of priming PCR amplification
selectively from DNA which contains the sequence that is targeted, such as
a point mutation, insertion, deletion, or the like.
In a broad sense, therefore, the present invention is directed to a method
for detecting the presence or absence of a target DNA sequence within an
identified region of a selected DNA molecule, wherein when the target is
present in the region, it has an expected location and configuration
therein. In the context of the present invention, the term "target DNA
sequence" is intended to refer generally, but not exclusively, to
variations, mutations and polymorphics. However, "target DNA sequences"
generally refers to any sequence present in a selected DNA molecule
wherein one desires to determine whether such a sequence is present in the
selected molecule, such as in comparison to a reference DNA molecule.
In certain embodiments, the identified region of the selected DNA molecule
will be a gene which is known or thought to include a particular mutation,
such as a point mutation. The mutation that is targeted for analysis, when
present in the gene, will have an expected location and configuration
therein. By this is meant that the mutation will generally, but not
always, have a known sequence, and will be present at a known location
within the gene. Accordingly, by "configuration" is meant that the
location of the mutation or target sequence, but not necessarily its
sequence, known with respect to the surrounding sequences of the gene or
other identified DNA region.
An example of such mutations which have been characterized to have an
expected location and configuration are seen in diseases such as diabetes,
sickle cell anemia, alpha and beta thalassemia, phenylketonuria,
hemophilia, alpha 1-antitrypsin deficiency, acute intermittent porphyria,
and possibly even diseases such as cystic fibrosis and Huntington's
chorea. Furthermore, cancer oncogenes such as N-ras, K-ras, H-ras, Neu, or
tumor suppressor genes such as p53 are known to have such mutations which
contribute to the cancer development. In the case of sickle cell disease,
the mutation involves the replacement of the sixth amino acid glutamate
with valine through an adenine to thymine nucleotide switch. Similar
mutations having expected locations and configurations are known in the
art and are applicable to the present invention, and the present invention
is applicable to such other mutations. Furthermore, additional mutations
having expected locations and configurations/will surely be characterized
in time, and the present invention will be likewise applicable to such
mutation as may be identified in the future.
As noted, the present invention involves an improvement to the PCR
amplification technique. As is known in the art, PCR amplification
involves generally the use of two separate primers which are capable of
binding along the 3' regions of the DNA region to which one desires to
amplify. While the present invention is preferably performed using two
separate primers, this is not an absolute requirement in that through the
practice of the invention one is attempting to determine whether the
primers are capable of priming DNA synthesis from a selected DNA molecule.
Accordingly, a first step of the present invention involves obtaining a
nucleic acid primer molecule having a template binding region that is
capable of hybridizing to a first strand of the selected DNA molecule at a
binding position 3' of and adjacent to the expected location of such a
target sequence within the DNA molecule. Thus, such primer molecules of
the present invention have the ability to bind at a position of the DNA
that is just 3' of the target sequence.
A novel aspect of the present invention, however, is that the primer
molecule further includes at its 3' terminus and at a position adjacent to
template binding region, a "target sequence complementary region"
comprised of at least one nucleotide that is complementary to a
corresponding nucleotide of the target sequence. Of course, where the
sequence that is targeted is a point mutation, the "target sequence
complementary region" will be a single nucleotide that is complementary to
the point mutation. Thus, where a point mutation involves an adenine, or
"A" residue, the primer molecule will include a thymidine or "T" residue,
at its 3' terminus. Where longer insertions, deletions or alterations are
to be detected, one may desire to include longer "target sequence
complementary regions", such as, for example, one, two or even 3 or more
nucleotides that are complementary to the targeted sequence. However, even
in these embodiments, only a single nucleotide that is complementary to
its corresponding nucleotide of the target sequence is required for
successful practice of the invention.
A unique and even surprising aspect of the invention involves the
inventors' finding that primers such as the foregoing are capable of
priming selectively only on sequences which include the targeted mutation.
Thus, where the selected DNA molecule does not include the target DNA
sequence within the expected location and configuration, the primer will
not function to prime DNA synthesis. Accordingly, a second step of the
invention in its broadest sense involves determining the ability of the
primer molecule to prime polymerase chain extension using the selected DNA
molecules template, to detect the presence or absence of the target
sequence within the selected DNA molecule.
In certain particular embodiments, the first nucleic acid primer molecule
will include, at a position one to three nucleotides 5, of the target
sequence complementary region, and positioned between the template binding
region and the target sequence complementary region, a nonsense nucleotide
on the selected DNA molecule. The inclusion of such a nonsense nucleotide
into the primer sequence at a position between the template binding region
and the target sequence complementary region provides for additional
assurances that the first primer will not effectively prime DNA synthesis
on templates which do not include a target sequence. That is, the
inventors have found that the inclusion of a nonsense nucleotide will not
appreciably inhibit the priming event so long as the 3' terminal
nucleotide is complementary to the template. However, the inclusion of
such a nonsense nucleotide provides additional advantages in that where
the 3' terminal nucleotide is not complementary to its corresponding
nucleotide along the template, the inclusion of the nonsense nucleotide
provides an additive effect in terms of preventing the occurrence of an
inadvertent priming event.
As noted, while one may employ simply a single primer such as the foregoing
to detect the presence of the target sequence within the selected DNA
molecule, one will generally desire to employ a second primer capable of
priming synthesis on the second strand of the selected DNA molecule, so as
to achieve PCR amplification of the region bearing the target, when such a
target is present therein. Accordingly, in certain embodiments,
determining the ability of the primer molecule to prime polymerase chain
extension will include obtaining a second nucleic acid primer molecule
having a second template binding region that is capable of hybridizing to
the second strand of the selected DNA molecule at a binding position 3' of
the expected location of such a target sequence, and determining the
ability of the first and second primers to prime polymerase chain reaction
synthesis of both strands of the DNA molecule, such an ability being
indicative of the presence of the target sequence within the selected DNA
molecule.
The second binding position on the second strand of the selected DNA
molecule can be located at virtually any binding position 3' of the
expected location of the target, so long as the chain extension can
proceed from the first binding position to the second binding position,
thus allowing PCR amplification. In that it has generally been found that
the enzyme currently in use for PCR amplification, the Taq enzyme, can
proceed up to 1,000 nucleotides, and perhaps even more, one could
theoretically choose a second binding position that is up to 1,000
nucleotides removed from the first binding position. However, this will
not generally be a preferred embodiment. Preferably, one will choose a
second binding position that is 30 to 600, and more preferably 60 to 260,
nucleotides removed from the first binding position.
In particular embodiments, determining the ability of the first and second
primers to prime polymerase chain reaction synthesis along with selected
DNA molecule will include steps of hybridizing the primers with the
selected DNA molecule to form primed templates, subjecting the primed
templates to polymerase chain extension to form polymerase chain extended
products, and detecting the generation of polymerase chain extended
products having a size corresponding to about the accumulative size of the
first and second primers together with the distance between their
respective primer binding positions along the DNA molecule. Thus, where
primers of about 20 to 40 nucleotides in length are employed, and separate
by about 100 nucleotides, the expected polymerase chain extension product
will have a size of about 140 to about 180nucleotides.
Of course, it will be appreciated that the primers may include DNA sequence
regions located 5' of the binding region which will not take part in
binding of the primer to the template, and such regions will be included
in the calculations of the expected size of the PCR extended product.
In certain embodiments, the generation of polymerase chain extended
products will be detected by subjecting the polymerase chain products to
gel electrophoresis, such as agarose gel electrophoresis, and identifying
products of the appropriate size, for example, by means of a label such as
a radionuclide, fluorescence, or enzyme. However, where several cycles of
synthesis are employed, it will generally be the case that the products
can be detected directly by simply staining the gel with appropriate DNA
staining reagents such as ethidium bromide.
While it is believed that one could successfully determine the ability of
the first and second primers to prime PCR synthesis, for example, through
the hybridization or use of radiolabeled nucleotides, one will generally
desire to employ several cycles of synthesis, such as, 10 to 60 cycles,
and more preferably 25 cycles of PCR synthesis.
In still further embodiments of the invention, one will desire to also
determine the ability of the primer molecules to prime polymerase chain
extension using a reference DNA molecule as template, wherein the
reference molecule includes an identified region which has been
characterized in terms of the presence of absence of the target sequence.
Thus the reference DNA molecule will generally be DNA, such as genomic
DNA, which includes the identified region, or gene, in a non-mutated form
(e.g., a sequence known not to include the point mutation, insertion,
deletion, etc.). Thus, such a reference molecule provides just that, a
reference against which one can measure the priming activity of the
unknown, selected DNA molecule. Where the selected DNA molecule is genomic
DNA obtained from a patient suspected of having agenetic disease, the
reference DNA molecule will generally be genomic DNA from an individual
known not to have the mutation being targeted by the assay. In such
embodiments, one will generally desire to simply compare the ability of
the respective first and second primers to prime synthesis on the selected
DNA in relation to the reference DNA.
However, in a still further aspect, one may desire to employ additionally
further primers designed to prime synthesis selectively on the reference
molecule and not on the selected molecule, if such a selected molecule
does not contain the target sequence. In such embodiments, the method of
the invention will further include obtaining a third nucleic acid primer
molecule having a third template binding region that is capable of
hybridizing to the first or second strand of the selected DNA molecule at
a binding position 3' of and adjacent to the expected location of such a
target sequence within the DNA molecule, the primer molecule further
including at its 3' terminus at least one nucleotide complementary to the
corresponding nucleotide on the reference DNA molecule.
Thus, rather than including at its 3' terminus a nucleotide complementary
to the target sequence, as is the case in the first primer molecule, the
third primer molecule will incorporate a 3' terminal nucleotide or
nucleotides which are complementary to corresponding nucleotides which
will be present when the target sequence is absent. Typically, this will
mean that the third nucleic acid primer molecule will have a 3' terminal
nucleotide(s) which is complementary to corresponding nucleotides of
non-mutated or non-variant sequences.
In accordance with the invention, in contrast to the first primer, the
third nucleic acid primer will prime synthesis on DNA molecules which do
not include the target sequence, such as a reference DNA molecule, and
will not prime synthesis on DNA molecules which have the target sequence.
Accordingly, the third nucleic acid primer molecules will then be used to
determine the ability to prime polymerase chain extension using either the
selected or referenced molecules template, such an ability being
indicative of the absence of the target sequence in the respective DNA
molecule, whether it be the reference or selected molecule.
In certain embodiments, the third template binding region will simply
correspond to the first template binding region, the of the third primer
only difference between the first and third primers being the identity of
their respective 3' terminal nucleotide or nucleotides.
Accordingly, as in the case of the first primer, one will generally desire
to employ a fourth primer for use in connection with the third primer in
order to determine the ability of the third primer molecule to prime
polymerase chain extension along with the selected or reference molecule.
In these embodiments a fourth nucleic acid primer is obtained having a
fourth template binding region that is capable of hybridizing to the
opposite strand from the third primer molecule at a binding position 3' of
the expected location of such a target sequence, and determining the
ability of the third and fourth primers to prime polymerase chain
extension on both strands of the reference or selected DNA molecule, such
an ability being indicative of the absence of the target sequence within
their respective DNA molecule. For convenience, the fourth nucleic acid
primer molecule can simply correspond or be equivalent to the second
nucleic acid primer molecule, when the first and third parimers recognize
the same strand.
As noted above, the present invention will have particular applicability to
the detection of point mutations in DNA molecules. Thus, in certain
embodiments, the present invention is directed to a method for detecting
the presence of a point mutation within a selected gene of a selected DNA
molecule wherein when said point mutation is present in the gene, it has
an expected location and configuration therein. The method of this aspect
of the invention will generally comprise the steps of (1) obtaining a
nucleic acid primer molecule having a template binding region that is
capable of hybridizing to a first strand of the selected DNA molecule at a
binding position 3' of and adjacent to the expected location of the point
mutation, the primer molecule further including at its 3' terminus and
adjacent to the binding region a nucleotide that is complementary to the
point mutation, and (2) determining the ability of the primer molecule to
prime polymerase chain extension using the selected DNA molecule template,
to detect the presence or absence of the point mutation within the
selected DNA molecule.
In still further aspects, the invention concerns novel nucleic acid primer
molecules for use in connection with polymerase chain extension for
determining the presence or absence of a DNA sequence that has an expected
location and configuration with an identified region of a selected DNA
molecule. The novel nucleic acid primer of the invention has a template
binding region that is capable of hybridizing to a first strand of the
selected DNA molecule at a binding position 3' of and adjacent to the
expected location of such a target sequence within the DNA molecule, the
primer molecule further including at its 3' terminus and adjacent the
template binding region a target sequence complementary region comprised
of at least one nucleotide complementary region to a corresponding
nucleotide of the target sequence.
In certain embodiments, the primer molecule further includes, at a position
one to three nucleotides 5' of the target sequence complementary region,
and positioned between the template binding region and the target sequence
complementary region, a nonsense nucleotide that is not complementary to
its corresponding nucleotides on the selected DNA molecule.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-C depict the PCR in vitro amplification of DNA through the use of
primers prepared in accordance with the present invention. The DNA
depicted in FIGS. A-C includes a sequence variant that is detected through
the practice of the invention. In FIG. 1A, the primers are depicted in
their hybridized position on a hypothetical template which includes the
variant. Depicted in FIG. 1B is the primed first cycle synthesis employing
the original mutated DNA molecule as the respective template for the
primers. In FIG. 1C is shown a second round of synthesis wherein elongated
chains from the first cycle of synthesis are reacted with additional
primers and polymerase enzyme to achieve a second cycle of synthesis with
the first cycle elongated chains serving as templates.
Shown in FIG. 2 is the application of the same primer shown in FIG. 1 to a
DNA molecule which does not include the mutation. As is depicted in FIG.
2, the primers are unable to achieve amplification of the desired region.
FIGS. 3A-B depict the preparation of primers that will specifically prime
synthesis on a wild type molecule which does not incorporate the target
sequence (FIG. 3A) as well as application of the wild type-directed
primers to a selected DNA molecule that does incorporate the target
sequence (FIG. 3B). As is depicted in FIG. 3B, the wild type-directed
primers are unable to support amplification of the desired region of the
selected primer which bears the target sequence.
Shown in FIGS. 4A-D is the application of primers to determine the presence
of absence of a point mutation in a selected DNA or will type DNA
molecule. Also shown in FIGS. 4C-D is the incorporation of a nonsense
nucleotide which provides further advantages in accordance with the
invention.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates generally to the preparation and use of
nucleic acid primers in the detection of sequence variations, mutations
and the like, in DNA samples. Primers are DNA molecules which are employed
to "prime" the synthesis or copying of a "template" DNA strand by a DNA
polymerase enzyme into a complementary strand. The newly generated
complementary strand will elongate from the primer into a new strand that
remains bound or hybridized to the template strand unless denatured.
In nature, DNA polymerases are required in order to catalyze DNA synthesis
prior to cell division, providing an "extra", exact copy or replica of a
cell's genomic DNA molecule to the daughter cells. The cell's entire DNA
complement is copied prior to cell division, through copying of each
strand into a complementary strand. Each DNA strand has a defined
polarity, a "3' direction" and a"5' direction", governed by the
head-to-tail arrangement of the pentose-phosphate backbone. The ribose
molecule portion of the pentose-phosphate backbone has a 5' carbon d a 3'
carbon linked to adjacent ribose molecules through aphosphate molecule.
Furthermore, complementary DNA strands run in an "anti-parallel" direction
with respect to each other, with one strand running in an opposite
direction of its complement. Thus, a 3' direction on one strand
corresponds to a 5' direction on the complement.
In order to achieve the enzymatic copying of a DNA strand, whether in a
cell (in vivo) or in a test tube (in vitro), the DNA polymerase enzyme
must have a starting point from which to begin its synthesis. This
starting point is the 3' terminus of the "primer" or "priming strand". The
primer is annealed to the template strand at a position at which DNA
synthesis begins. During DNA replication, the DNA polymerase enzyme begins
its copying of the template strand at the 3' end of the priming strand and
forms a covalent phosphate linkage with the 5' carbon of the growing
chain. Due to the fact that DNA replication has a 3' to 5' polarity of
elongation, synthesis proceeds in a 3' direction with respect to the
strand that is being copied (the template).
The present invention relies in part on the observation that in order for a
primer molecule to successfully "prime" DNA synthesis from a template, the
3'-terminal nucleotide of the primer must be capable of base pairing with
the template. That is, the 3'-terminal nucleotide of the primer must, in
essence, be capable of "lying flat" against the template strands and
remain in a proper configuration for a sufficient length of time to
achieve a priming of the DNA polymerase enzyme's ability to proceed with
synthesis. Thus, if a particular DNA molecule that one attempts to employ
as a primer is otherwise capable of annealing to the template, but does
not have a 3'-terminal nucleotide that can "lie flat" against the template
for a sufficient period of time, the DNA molecule cannot properly function
as a primer. Such a "primer" molecule which does not have a 3'-terminal
nucleotide that is capable of hybridizing with or otherwise "lying flat"
against the template strand is said to have a "3'-terminal flap" or a
3,-terminus which "flaps away" from the template.
The present invention takes advantage of this observation to provide a
simple and reliable means for detecting the presence or absence of a
particular DNA target sequence within a gene or other region of a selected
DNA molecule. In a general sense, this is achieved by providing a primer
molecule that will successfully prime DNA chain elongation only on DNA
template molecules to which it can both hybridize and having a 3' terminus
that will base pair with or otherwise lie flat against the template (i.e.,
where the template contains a nucleotide that is complementary to the 3'
pair with the 3' terminus of the primer when the primer is annealed to the
template).
When these conditions are met, the primer will successfully prime chain
elongation. However, if the template does not include a complementary
nucleotide in the 3' terminal position, the primer will not prime chain
elongation. This provides the ability to prepare primers that will
successfully prime chain synthesis only where the template strand contains
a particular expected variant or mutation, but will not prime synthesis on
control or "wild-type" templates that do not include the mutation.
Alternatively, it provides the ability to prepare primers that will prime
chain elongation only of templates that do not include the mutation or
variant, and will not prime synthesis of templates that include the
variant or mutation.
The only requirement for preparing and employing primers of the present
invention, is that one must know the approximate sequence of the region of
the template strand that one is seeking to investigate, as well as the
expected location or "configuration" of the variant or mutation "target,
when it is present. This knowledge allows one to prepare primers that will
hybridize and prime only under the selected conditions. While not
absolutely necessary it will be desirable to know the expected nucleotide
sequence of the variant or mutation. This allows one to employ a single
primer that can be used universally, e.g., where one is seeking to develop
an assay for screening for a particular mutation or genetic abnormality.
Of course, while many genetic diseases involve mutations that occur at an
expected site within a gene, it is not always the case that such mutations
will involve the same nucleotide cha | | |
