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Description  |
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This invention relates to methods and products useful for detecting the
presence or absence of a particular nucleotide at a specific location on a
strand of DNA. By using the methods and products of this invention, it is
possible to determine the genotype of an individual at any locus of
interest.
A single nucleotide position on a strand of DNA may be responsible for
polymorphism or allelic variation. There are known disease states that are
caused by such variation at a single nucleotide position. The usefulness
of detecting such variation includes but is not limited to gene typing,
karyotyping, genotyping, DNA family planning, diagnostics (including
infectious disease), prenatal testing, determining parentage, and forensic
analysis.
The typical methods for determining such variation have been to hybridize
specific probes to permit Southern Blots containing different DNA digests
to test for variation in the length of specific restriction fragments, or
to amplify specific regions of DNA samples by polymerase chain reaction
(PCR), and test for nucleotide variation by sequence analysis or by
hybridization with allele specific probes.
Each of these methods has certain drawbacks, including: the lack of
reproducibility of Southern analysis, the need for running gels to
separate DNA fragments, and the extended amount of time required to
complete the necessary steps in the process. PCR techniques suffer from
occurrence of false signals arising from contamination, and the time and
technical expertise required for the determination of sequences from PCR
amplified samples. However, perhaps the most serious drawback is that both
methods require a number of separate analyses to test for variation at
more than one DNA locus.
SUMMARY OF THE INVENTION
The present invention involves a novel technique for determining the
existence or nonexistence of a particular nucleotide at a particular
position on a strand of DNA. The determination requires the use of a
special primer capable of pairing with the strand of DNA and carrying a
detectable label which will be retained if there is a base pair match at
the nucleotide of interest (i.e., the test nucleotide), but will be lost
if there is a mismatch at the nucleotide of interest. The determination
advantageously employs an exonucleolytic agent that will remove the label
if there is a base pair mismatch at the nucleotide of interest when the
labeled primer and DNA are paired with one another.
According to one aspect of the invention, a test sample of DNA is treated
with a labeled oligonucleotide primer of the invention, the primer being
capable of pairing with a first portion of the DNA adjacent to the test
nucleotide. The primer of the invention includes a labeled nucleotide at
or near the position opposite to the test nucleotide when the primer and
the DNA are paired. Then, the primer-DNA pair is subjected to conditions
that allow retention of the labeled nucleotide attached to the primer if
there is a base-pair match at the test nucleotide position, but not if
there is a mismatch. Then, the presence or absence of the label on either
the excised free base or on the extended primer is determined.
The method of the invention employs an agent which is capable of excising
the label in the primer if there is a base pair mismatch at the nucleotide
of interest when the primer and DNA are paired with one another. The agent
preferably also includes polymerase activity as well as exonuclease
activity, although polymerase activity is nonessential. The important
consideration is that conditions be applied to the paired primer-DNA
strand such that the label in the primer will be retained in the presence
of a base pair match at the test position, but not in the presence of a
base pair mismatch at the test position.
The invention has the advantage of not requiring the time-consuming and
troublesome gels employed in certain of the prior art detection
techniques.
The presence or absence of retained label attached to the primer may be
detected in various ways. For example, loss of label from the primer could
be measured by detecting the presence of label unassociated with the
primer. Any free label could be separated from the larger primers and
sample DNA molecules by methods such as ultrafiltration, gel filtration,
HPLC, mass spectrometry or capillary zone electrophoresis. Detection of
the presence of free label would indicate that a mismatch had occurred in
the test and that the exonucleolytic agent had removed the label from the
primer.
Alternatively, the primers may be extended by the action of a polymerase in
the presence of an exonucleolytic agent. A match or mismatch then may be
detected by determining the presence or absence of label on the extension
product. Such a determination is well within the knowledge of one of
ordinary skill in the art, and may be accomplished, for example, by
hybridizing the extension product to its complement fixed to a solid
support and determining the presence or absence of label.
In another approach, the presence or absence of label in the primer
extension product is determined according to a method that requires an
oligonucleotide primer having attached to it a unique tail sequence
non-complementary with the DNA. The primer extension product then is
applied to a substrate carrying an oligonucleotide, at least in part
complementary to the unique tail sequence, of the extension product, under
conditions that allow the unique tail sequence to hybridize to the
complementary oligonucleotide on the substrate. Preferably, both the tail
and the oligonucleotide complementary to the tail are comprised of
repeating units of complementarity. This favorably affects the kinetics of
hybridization, increasing the speed and the sensitivity of the test. Of
course, unreacted primer must be minimized or removed prior to the assay
phase of this approach to prevent it from hybridizing to the substrate and
interfering with The interpretation of the results.
In still another approach, the presence or absence of the mismatch may be
determined in solution or on a solid support by using nonradiative
fluorescent resonance energy transfer (FRET, see Cardullo et al., 1988,
Proc. Nat'l. Acad. Sci. USA 85: 8790-8794) between two suitable
fluorescent labels. With this technique, the labels would allow effective
FRET only if both fluorescent labels were maintained in close proximity.
In this approach, two labels could either be provided in adjacent
positions on the primer, or one could be on the primer and the other
incorporated via intercalation into the duplex DNA formed by the primer
and the test DNA strand. A mismatch would interfere with measurable FRET
by allowing exonucleolytic excision of one or both of the fluorescent
labels.
The invention allows for determining genotype at desired genetic loci. The
test DNA is exposed to a plurality of different oligonucleotide primers,
each of the primers being complementary to a genetic locus of interest.
Each primer carries a labeled nucleotide at or near one end, said labeled
nucleotide also being at or near the position opposite the test nucleotide
(which is known to vary in different alleles of the locus) when the primer
and test DNA are paired. The treatment includes subjecting the primers and
DNA to conditions that allow the primers and DNA to pair. The paired
primer-DNA then is treated with an exonucleolytic agent under conditions
that allow the retention of label in the primer if there is a match
between any test nucleotide and the complementary nucleotide on the primer
opposite to a test nucleotide, but loss of the label in the primer if
there is a mismatch. The presence or absence of label in a primer (which
corresponds to the presence or absence of an allelic complementation) then
may be determined by applying the samples containing the reaction
products, after denaturation, to substrates spotted at distinct locations
with unique oligonucleotides complementary to the
polymerase/exonuclease-catalyzed extension products. If label has been
retained in the primer extension product, then it will be detected at a
unique location on the substrate; the product attaches to the substrate at
only one location via the hybridization to the unique, complementary
oligonucleotide found only at that location. The presence or absence of a
specific allelic sequence thus may be determined by detecting the presence
or absence of label at a specific location on the substrate,
The foregoing test for genotype is particularly useful when determining
genotype for a single allele. For identification of multiple alleles, the
technique may be repeated using allele-specific primers on multiple
samples. Alternatively, multiple alleles may be assayed simultaneously
using primers of different allelic complementarity having allele-specific
labels or tails.
The invention thus provides an oligonucleotide primer having a sequence of
nucleotides complementary with at least a portion of a DNA strand and
terminating at or very close to a test nucleotide position on the DNA
strand, nucleotide variation at that test position being responsible for
polymorphism. The oligonucleotide primer has a labeled nucleotide at or
near the position which will be opposite the test nucleotide when the
primer and the test DNA are hybridized. Sets of such oligonucleotide
primers are provided for determining the genotype of an individual at
specific loci.
The invention further utilizes an agent with exonucleolytic activity
capable of excising the label on the primer of a paired primer-DNA strand
having a mismatched base-pair at or close to an end of said primer. This
exonucleolytic agent may have no polymerase activity. The exonucleolytic
agent may be a native exonuclease or polymerase with exonucleolytic
activity, a mutant exonuclease or polymerase with exonucleolytic activity,
or a non-mutant exonuclease or polymerase with exonucleolytic activity
treated to impart the necessary properties.
The invention also provides a substrate having attached to it at one
location a first oligonucleotide having a first sequence and at a second
location a second oligonucleotide having a sequence different from and
non-complementary with the first oligonucleotide. These substrate
oligonucleotides are complementary with the primers, their tails, or their
extension products. Preferably, the substrate is spotted with
oligonucleotides firmly bound to the substrate but accessible for
hybridization with complementary sequences. The substrate may have
attached to it in such a manner at known locations many different
oligonucleotides which are non-complementary with each other.
The products of the invention may be advantageously provided in kits. The
kits may include a plurality of different oligonucleotide primers and a
plurality of oligonucleotides complementary to portions of DNA extended by
the action of the polymerase/exonuclease of the invention on the primers
when paired to the test DNA. Most preferably, the kit includes a substrate
having attached at different locations the complementary oligonucleotides.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically shows the synthesis of an extension product with
retention of label obtained by using the exonuclease/polymerase of the
invention when there is a match at the test position;
FIG. 2 schematically illustrates the synthesis of an extension product with
loss of label obtained by using the exonuclease/polymerase of the
invention when there is a mismatch at the test position;
FIG. 3 schematically shows an oligonucleotide of one embodiment of the
invention, which primer includes a unique tail sequence;
FIG. 4 schematically shows the detection of an extension product formed
with retention of label on the oligonucleotide primer of FIG. 3;
FIG. 5 schematically shows a detection substrate for detecting the label on
the oligonucleotide primer of FIG. 3;
FIG. 6 schematically shows a set of preferred oligonucleotide primers;
FIG. 7 schematically shows a substrate for determining allelic variation;
FIG. 8 schematically shows a second set of oligonucleotide primers for
detecting multiple alleles at multiple loci;
FIG. 9 schematically shows a primer of the invention hybridized to phage
M13mp19(+) strand carrying a region complementary to the primer.
DETAILED DESCRIPTION OF THE DRAWINGS
The present invention involves a novel technique for determining the
existence or nonexistence of a particular nucleotide at a specific locus
on a strand of DNA, at least a portion of which strand has a known
sequence, adjacent to and including the locus of interest. The invention
may be used in connection with many medical tests, including all of those
listed in the background of the invention. It is particularly useful in
determining an individual's genotype at the test locus, especially as the
genotype relates to the existence of an allele or mutation responsible for
a disease state or as it relates to an individual's identity.
The invention involves using a labeled oligonucleotide primer that will
either create a base pair match or mismatch between a test nucleotide on
the DNA strand and the labeled nucleotide at the opposite position on the
primer when the primer is paired with the DNA strand. The primer is
labeled at only one or at a few position(s), that are near or at the
position opposite the test nucleotide. First, the primer and DNA strand
are caused to pair. Then, conditions are applied to the primer-DNA pair
that will cause retention of the label in the primer product in the
presence of a match, but not in the presence of a mismatch. The test
involves the use of an exonucleolytic agent that removes the label on the
primer if there is a mismatch when the primer and DNA are paired, but does
not remove the label on the primer if there is a match.
The term "oligonucleotide" as used herein in referring to primers,
extension products, tails and products complementary to primers, extension
products and tails, refers to a molecule comprised of two or preferably
more than three deoxyribonucleotides or ribonucleotides, synthetic or
natural. The exact size of the molecule may vary according to its
particular application.
The term "primer" as used herein refers to an oligonucleotide which, when
paired with a strand of DNA, is capable of initiating the synthesis of an
extension product in the presence of a suitable polymerization agent.
Preferably, the primer is an oligoribonucleotide and most preferably is an
oligodeoxyribonucleotide. The primer, however, may be other than a
ribonucleotide. The primer must be sufficiently long to hybridize uniquely
to the test region of the test DNA strand, and the primer must contain a
labeled nucleotide at or near the position opposite the test nucleotide of
the test DNA strand. The exact length of the primer will depend on many
factors, including the degree of specificity of pairing required, and the
temperature and ionic strength during hybridization.
The term "exonucleolytic agent" as used herein may be any compound or
system which will function to accomplish the removal of the label when the
appropriate nucleotide(s) are mismatched. Suitable enzymes for this
purpose may include DNA polymerases with exonucleolytic acitivity,
single-strand specific exonucleases, and other enzymes, including
heat-stable enzymes. Generally, the exonucleolytic agent will be a 3' to
5' exonuclease directed to the 3' end of each primer. There are also
agents, however, which remove nucleotides at the 5' end or both from the
5' and the 3' end, and these may also be used.
The term "terminal nucleotide" as used herein in referring to
oligonucleotide primers refers to the terminal nucleotide at either end of
the primer. When the primer is hybridized to the test DNA, the nucleotide
position opposite to the position of the test nucleotide on the DNA strand
is located at or close to a terminal nucleotide.
The term "pairing" as used herein contemplates any and all methods of
sequence specific pairing between the primer and a strand of DNA including
the pairing of a primer with double stranded DNA, so long as an
exonucleolytic agent may act on the product of such a pairing. Typically,
however, a single stranded primer and a single strand of DNA will be
paired by subjecting them to conditions which cause them to hybridize to
one another. The primers are selected to be "substantially" complementary
to the strands of each specific DNA sequence being tested. By
substantially it is meant that the primer is sufficiently complementary to
pair with the test DNA. The primer sequence then need not reflect the
exact sequence of the test DNA. However, in a preferred embodiment, the
primer is at least 20 nucleotides long and contains no mismatches with the
complementary DNA strand except in certain instances at or close to the
nucleotide position complementary to the test nucleotide.
The term "label" as used herein refers to, but is not limited to, the
following classes of reporter groups. Primary labels such as radioisotopes
and fluorescent groups are signal generating reporter groups which can be
detected without further modifications. Secondary labels such as biotin
and various protein antigens act as "bridges" and require the presence of
a second intermediate for production of a detectable signal. For biotin,
the secondary intermediate may include. streptavidin-enzyme conjugates.
For antigen labels, secondary intermediates may include antibody-enzyme
conjugates. Some fluorescent groups act as secondary labels because they
transfer energy to another group in the process of nonradiative
fluorescent resonance energy transfer, and the second group produces the
detected signal.
The terms "match" and "mismatch" refer to the hybridization potential of
paired nucleotides in complementary strands of DNA. Matched nucleotides
hybridize efficiently, such as the classical A-T and G-C base pairs.
Mismatches are other combinations of nucleotides which do not hybridize
efficiently.
Precursors to the labeled oligonucleotide primers (including tails) of the
invention or the oligonucleotide primers themselves may be prepared using
any suitable method, such as, for example, methods using phosphotriesters
and phosphodiesters well known to those skilled in the art. In one such
automated embodiment, diethylphosphoramidites are used as starting
materials and may be used for synthesis of oligonucleotides as described
by Beaucage and Caruthers, 1981, Tetrahedron Letters, 22: 1859-1862. One
method for synthesizing oligonucleotides on a modified solid support is
described in U.S. Pat. Nos. 4,458,066 and 4,500,707. It is also possible
to use a precursor primer or primer which has been isolated from a
biological source (such as a restriction endonuclease digest of plasmid or
phage DNA).
Labels may be applied to precursor primers by any suitable method including
enzymatic methods. For example, DNA polymerases such as the Klenow
fragment of DNA polymerase I will add certain labeled nucleotides to the
3' end of a precursor primer in the presence of the suitable template DNA
strand. Suitable labels include, but are not limited to, .sup.32 P,
biotin, and fluorescent moieties such as rhodamine or fluorescein.
According to a preferred embodiment of this invention, an agent for
polymerization having exonuclease activity is used to indicate whether a
primer contains a nucleotide which is complementary to, or not
complementary to, the test nucleotide in the DNA strand. Typically, a
single stranded primer when hybridized to a longer single strand of DNA in
the presence of nucleoside triphosphates and an agent for polymerization,
at suitable temperature and pH, will allow the synthesis of an
oligonucleotide attached to and extending from the primer, the
oligonucleotide being complementary with the single strand of DNA. Many
known agents of polymerization will not catalyze extension of a primer
from a mismatched terminal base pair. Rather, the agent of polymerization
will excise the mismatched base pair by its exonucleolytic activity and
then initiate the synthesis of an extension product from a matched pair.
As shown in FIG. 1, if there is complementary base pairing (a match)
between the labeled terminal nucleotide 12 of the primer 14 and the test
nucleotide 16 on the test DNA 18, an extension product will be synthesized
and the labeled nucleotide (*) will be retained. However, as shown in FIG.
2, if there is a mismatch between the labeled terminal nucleotide 12 of
the primer 14 and the test nucleotide 16, then an extension product will
be synthesized but only after excision of the mismatched labeled
nucleotide opposite the test position in the DNA strand.
The synthesis of the extension product may be according to methods
well-known to those skilled in the art. For example, if a
deoxyribonucleotide extension product is being synthesized, the hybridized
primer-DNA strand must be treated with a polymerase having exonucleolytic
activity in the presence of deoxyribonucleoside triphosphates (dATP, dCTP,
dGTP, dTTP). According to a preferred embodiment, the primer carries a
labeled nucleotide at or close to its 3' end. Typical labels include
.sup.32 P-labeled, biotin labeled, or fluorescent labeled nucleoside
triphosphates. The labelled nucleotide is at or near the position opposite
the test position on the test DNA strand when the primer is hybridized to
the test DNA strand.
By using a polymerase/exonuclease, which excises a mismatched nucleotide in
the primer before synthesizing an extension product, and by subjecting the
hybridized primer-DNA to conditions that permit excision and extension,
the presence or absence of a specific nucleotide on a strand of DNA may be
determined. For example, assume that a gene for a health trait includes
the following known sequence: 3'GATCGAATTGGCACACGTT5'. Also assume the
gene for the disease state is due to or correlated with a substitution at
a single test nucleotide position, underlined: 3'GATCGAATTGGCCCACGTT5'. A
primer that could be used to detect the presence or absence of the disease
state then would be: 5'CTAGCTTAACCGG*3', in which the terminal "G" is
labeled (the * denotes the label). This primer is capable of hybridizing
with either of the foregoing DNA sequences. However, when the primer
hybridizes to the DNA sequence characteristic of the healthy state, there
will be a mismatch at the terminal end of the primer, an A being paired
with a G. If that hybridized primer-DNA strand is treated with a
polymerase/exonuclease then an extension product will be formed, but only
after excision of the labeled "G" residue of the primer. On the other
hand, when the primer is hybridized with the DNA sequence characteristic
of the disease state, there is a match between the terminal nucleotide of
the primer and the test nucleotide on the DNA strand (G-C). That
hybridized primer-DNA strand will initiate the synthesis of an extension
product with retention of the labeled "G" residue in the presence of the
polymerase/exonuclease of the invention.
To determine whether a sample of test DNA carries the DNA characteristic of
the healthy state or the disease state, the labeled primer is added to a
sample of test DNA under conditions allowing the primer to hybridize to
the test DNA. A polymerase/exonuclease and nucleoside triphosphates then
are added and the mixture is subjected to conditions that allow excision
and synthesis of an extension product. It then is determined whether label
is present or absent in the resulting extension product. If label was
retained in the extension product, then there was a match indicating the
presence of the DNA characteristic of the disease state. If label was lost
in the extension product, then there was not a match and the nonexistence
of the DNA characteristic of the disease state is established.
Detection methods well known to those skilled in the art may be employed to
determine the presence or absence of label in the extension product. DNA
complementary to the extension product may be attached to a substrate such
as filter paper or a nylon membrane. This substrate may be treated with
the products of the test reaction under conditions that would allow any
extension product to hybridize to the complementary DNA on the substrate.
The substrate then would be tested for the presence or absence of any
label attached via hybridization to the complementary strand. If there is
label on the substrate, then the label was retained during synthesis of
the extension product. If there is no label on the substrate, then the
label was excised prior to synthesis of the extension product.
According to another embodiment of the invention, the test is improved by
using a primer 24 having primer portion 25 and a tail portion 26 attached
to and extending from the end of the primer portion 25 opposite the
labeled terminal nucleotide 12. Preferably, the tail portion 26 is unique
and is non-complementary with the test DNA. Such a primer is shown
schematically in FIG. 3 hybridized to a longer strand of DNA 18. When
using the labeled primer 24 of the invention and a polymerase/exonuclease,
an extension product 27, having three portions, is formed (FIG. 4). The
extension product will include the extension portion 28, the primer
portion 25, labeled or unlabeled depending on the test nucleotide, and the
tail portion 26.
Improvements to the speed and sensitivity of the assay are achieved using
such primers having tails. The presence or absence of label in the primer
portion of the extension product may be detected by using substrates such
as filter paper 30 spotted with a great excess of oligonucleotide
complementary to the tail portion 26. Because such complementary
oligonucleotide DNA 32 may be synthesized inexpensively in great quantity
and therefore may be applied to the substrate in great excess (FIG. 4),
the rate and amount of hybridization between the tail portion 26 of the
extension product 27 and the complementary oligonucleotide 32 on the
substrate is enhanced.
Most preferably, the oligonucleotide of the tail and the oligonucleotide
complementary to the tail both consist of repeating units of
complementation. Most preferably, the tail portion 26 is a polymer
consisting of repeating units of an oligonucleotide 14 nucleotides long,
and the complementary oligonucleotide 32 is a polymer consisting of
repeating units of an oligonucleotide that is also 14 nucleotides long.
The use of such repeating units of complementation favorably affects the
kinetics of hybridization, further increasing the speed and the
sensitivity of the assay.
A substrate having attached to it a plurality of polymers 33 of such
repeating units 34 of complementation is shown schematically in FIG. 5.
Preferably, the plurality of polymers 33 are covalently linked to the
substrate at a very high concentration to form a solid solution that
presents a great many available hybridization sites, unobstructed by the
substrate to which the polymers are attached. These substrates with
attached polymers may be dried out and stored for considerable periods.
The products and methods of the invention may be used advantageously to
determine allelic variation in genotyping studies. For example, if allelic
variation is due to a single nucleotide substitution (or is correlated
with such a substitution), then test DNA can be treated using primers for
both alleles to determine whether an individual is homozygous or
heterozygous with respect to those alleles. Such a test is performed
advantageously using primers for each allele having tails differing from
one another so that only a single test carried out in a single vessel is
necessary.
To accomplish this, two primers are constructed as shown in FIG. 6. Each
primer has a primer portion P that is complementary to the same DNA,
except that the labeled terminal nucleotide on each of the primers is
different. The labeled terminal nucleotide on one of the primers is
complementary to the nucleotide determining one allele and the labeled
terminal nucleotide on the other primer is complementary to the nucleotide
determining the second allele. In the example shown, the labelled terminal
nucleotides are cytosine and adenosine (C and A, respectively).
At the opposite end of each of the primers is attached a unique tail. By
"unique" it is meant that a sequence complementary to one tail will not
hybridize with the other tail. Moreover, neither of the tails and neither
sequence complementary to the tails should be capable of hybridizing with
the test DNA. It is believed that a single nucleotide substitution on an
oligonucleotide 14 nucleotides long is sufficient to prevent cross
hybridization. Preferably there are at least two nucleotide substitutions
to distinguish each tail. As is understood by those skilled in the art,
the synthesis of a set of thousands of such unique tails 14 nucleotides
long is possible.
The designations for the primers shown in FIG. 6 are T.sub.1 P.sub.1 C* and
T.sub.2 P.sub.1 A*: the T signifying tail and the subdesignation
signifying the sequence of the tail; the P signifying primer portion and
the subdesignation signifying the sequence of the primer; and the last
letter signifying the labeled terminal nucleotide. Thus, T.sub.1 P.sub.1
C* stands for tail sequence number 1, primer sequence number 1, and a
cytosine terminal nucleotide. T.sub.3 P.sub.2 A* would stand for tail
sequence number 3, primer sequence number 2 and adenosine as a terminal
nucleotide.
The primers shown in FIG. 6 (T.sub.1 P.sub.1 C* and T.sub.2 P.sub.1 A*) are
added to test DNA under conditions that allow the primers to hybridize
with the test DNA. Then the hybridized primer-DNA may be treated with a
polymerase/exonuclease and nucleoside triphosphates under conditions that
allow the synthesis of an extension product with retention of label if
there is a match at the labeled terminal nucleotide. Thus, if the test DNA
has a G at the test nucleotide, which is complementary to the labelled
terminal nucleotide of the primer T.sub.1 P.sub.1 C*, then there is a
match and an extension product will be synthesized with retention of the
label. Likewise, if the test DNA has a T at the test nucleotide which is
complementary to the terminal nucleotide of the primer T.sub.2 P.sub.1 A*,
then there is a match and an extension product will be synthesized, with
retention of the label. The sample containing the extension products then
is applied to a substrate having spotted at different locations an
oligonucleotide complementary to tail number 1 (T.sub.1 ') and an
oligonucleotide complementary to tail number 2 (T.sub.2 ') (FIG. 7).
Extension product will hybridize at T.sub.1 ' via hybridization of tail
number 1 to the T.sub.1 ' oligonucleotide and extension product also will
hybridize to spot T.sub.2 ' via hybridization of tail number 2 to the
oligonucleotide at T.sub.2 '. The presence of label at both locations
would indicate a heterozygous individual. If, on the other hand, label is
detected only at spot T.sub.1 ', then the individual carries only a G at
the test nucleotide. Likewise, if label is only detected at spot T.sub.2
', then the individual carries only a T at the test nucleotide position.
Thus, the genotype of an individual at a single locus may be determined in
a single test, two alleles being tested for simultaneously.
It will be understood by those skilled in the art that the genotype could
have been tested by using primers having the same tail, rather than unique
tails. To accomplish this, the primers must be tested separately with
separate samples of test DNA. It, however, is an advantage of the
invention that by using unique tails, any number of alleles or loci may be
tested for simultaneously. Thus, tests for different genes and tests for
multiple alleles on different genes may be accomplished simultaneously
according to the invention. For example, a plurality of primers may be
constructed, including primers complementary to different genes. FIG. 8
depicts a set of primers for three genes, each gene having two alleles.
T.sub.1 P.sub.1 C* and T.sub.2 P.sub.1 G* are complementary to the same
gene, but to different alleles; T.sub.3 P.sub.2 A* and T.sub.4 P.sub.2 G*
are complementary to the same second gene, but to different alleles; and
T.sub.5 P.sub.3 C* and T.sub.6 P.sub.3 G* are complementary to a third
gene, but also to different alleles of that gene. Each of the primers has
a unique tail (T.sub.1, T.sub.2, T.sub.3, T.sub.4, T.sub.5, and T.sub.6),
and the terminal nuclotide of each primer is labelled. When this set of
primers is mixed with a single sample of test DNA, only those primers that
have hybridized to the test DNA and have matching nucleotides at the
terminal end of the primer are capable of initiating the synthesis of an
extension product retaining the labeled nucleotide. After the conditions
for the exonuclease/polymerase reaction have been applied, the label on
any unreacted primers may be removed by use of a potent 3' to 5'
exonuclease activity such as that of T4 DNA polymerase, which prefers
single-stranded DNA. Alternativly, label on any unreacted primers may be
removed by separating extension products from unreacted primers. Next, the
products of the reaction may be placed in contact with specific
oligonucleotides, complementary to the unique tails, spotted at different
locations on a substrate. Then, the existence of label on the reacted
primers is determined by looking for the presence of label on the
substrate, potentially present due to reacted primers hybridizing via
their tails to the substrate. The existence of label at a particular
location on the substrate indicates that label was retained on the primer
portion of an extension product, the primer being identified by its unique
tail complementary only with the oligonucleotide at the particular
location. Thus, the presence or absence of each of the various genes and
multiple alleles may be tested simultaneously using a single sample of
test DNA.
For the implementation of these first two embodiments, the complementary
DNA attached to the substrate may be complementary to at least one of the
following: a portion of the primer (including complementation to only the
tail portion), a portion of the synthesized extension product, or a
portion of both. If the complementary DNA on the substrate is
complementary to a portion of the primer, it would be necessary to remove
nonhybridized, labelled primers from the reaction mixture prior to contact
of the mixture with the substrate-bound oligonucleotides. Otherwise, the
presence of label on the substrate might not be the result of a match
between the labeled nucleotide and the test nucleotide, but might simply
result from the presence of primer which failed to hybridize. This could
be accomplished in a variety of ways including: ensuring that most of the
labelled primer molecules had an opportunity to hybridize to the test DNA
and undergo reaction with a polymerase/exonuclease; treating the unreacted
primers with a potent exonuclease preferring single stranded DNA to excise
the labelled terminal nucleotide; or alternatively, removing unreacted
primer molecules from the solution containing the extension product.
In order to ensure that most of the labeled primers participate in the
reaction of the invention, it is helpful to repeat the primer annealing
and exonucleolytic phases of the reaction several times. For example, the
reaction may be heated to dissociate hybridized extended primer and test
DNA and then cooled to permit annealing of new primers to the test DNA. If
the polymerase/exonuclease used is not heat stable, then more would be
added, and the reaction mixture incubated under conditions to permit
exonucleolytic action and polymerization. The entire cycle is repeated
until calculations indicate that most of the added labeled primer had
participated in the reaction. Alternatively, a control primer is included
in a separate similar reaction. The control primer carries a labeled
terminal nucleotide which is not complementary to the corresponding
nucleotide in any of the alleles in question. Loss of most of the label in
the control reaction indicates that sufficient cycles were carried out to
permit most of the added labeled primers to participate in the react | | |
