U.S. patent application number 09/817815 was filed with the patent office on 2001-09-06 for method for amplification of dna.
This patent application is currently assigned to One Lambda. Invention is credited to Blair, Lindley, Lee, Jar-How.
Application Number | 20010019825 09/817815 |
Document ID | / |
Family ID | 22538890 |
Filed Date | 2001-09-06 |
United States Patent
Application |
20010019825 |
Kind Code |
A1 |
Lee, Jar-How ; et
al. |
September 6, 2001 |
Method for amplification of DNA
Abstract
The invention provides methods for detecting target nucleic acid
sequences with diagnostic primers including priming regions and
probe regions which are complementary to target and reference
regions respectively on a sample nucleic acid strand wherein the
probe region is located 5' to the priming region which is
complementary to a reference nucleic acid sequence which is 3' to
the target nucleic acid sequence on the sample nucleic acid strand
wherein when said reference nucleic acid sequence is contiguous
with said target nucleic acid sequence on the sample nucleic acid
strand then the priming region and probe region on the diagnostic
probe are separated by a spacer region of nucleic acid.
Inventors: |
Lee, Jar-How; (Los Angeles,
CA) ; Blair, Lindley; (Los Angeles, CA) |
Correspondence
Address: |
MARSHALL, O'TOOLE, GERSTEIN, MURRAY & BORUN
6300 SEARS TOWER
233 SOUTH WACKER DRIVE
CHICAGO
IL
60606-6402
US
|
Assignee: |
One Lambda
|
Family ID: |
22538890 |
Appl. No.: |
09/817815 |
Filed: |
March 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09817815 |
Mar 26, 2001 |
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09151465 |
Sep 11, 1998 |
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6207379 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 536/24.3 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6858 20130101; C12Q 1/6858 20130101; C12Q 1/6881 20130101;
C12Q 2537/143 20130101 |
Class at
Publication: |
435/6 ;
536/24.3 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A method for detecting the presence of a target nucleic acid
sequence on a sample nucleic acid strand from an individual
comprising the steps of: treating the sample, together or
sequentially with appropriate nucleoside triphosphates, an agent
for polymerization of the nucleoside triphosphates, a diagnostic
primer and an amplification primer under hybridizing conditions;
wherein the nucleotide sequence of said diagnostic primer comprises
(1) a priming region at its 3'-end which is substantially
complementary to the target nucleic acid sequence, and (2) a probe
region located 5' to said priming region which is substantially
complementary to a reference nucleic acid sequence which is 3' to
the target nucleic acid sequence on the sample nucleic acid strand
wherein when said reference nucleic acid sequence is contiguous
with said target nucleic acid sequence on the sample nucleic acid
strand then the priming region and probe region on the diagnostic
probe are separated by a spacer region of nucleic acid, whereby for
selected hybridization and extension conditions an extension
product of the diagnostic primer is synthesized when the priming
region is substantially complementary to the target nucleic acid
sequence and when the probe region is substantially complementary
to the reference nucleic acid sequence, but wherein for said
selected hybridization and extension conditions no extension
product of the diagnostic primer is synthesized when either the
priming region or the probe region are not substantially
complementary to the target and reference nucleic acid sequences
respectively; any extension product of the diagnostic primer formed
being capable of serving as a template for synthesis of an
extension product of said amplification primer after separation
from its complement; amplifying any extension product; and
detecting the presence or absence of the target polynucleic acid
sequence from the presence of absence of amplification product
obtained as above.
2. The method of claim 1 wherein said priming region and said probe
region on the diagnostic primer are separated by a spacer region of
nucleic acid sequence which is not complementary to the sequence of
the sample nucleic acid strand between the target and reference
sequences
3. The method of claim 2 wherein said spacer is from 1 to 30 bases
long.
4. The method of claim 2 wherein said spacer is from 8 to 30 bases
long.
5. The method of claim 1 wherein said target nucleic acid sequence
is from 1 to 350 bases from said reference nucleic acid sequence on
a sample nucleic acid sequence.
6. The method of claim 1 wherein the priming region and probe
regions are exactly complementary to the target and reference
nucleic acid sequences respectively.
7. The method of claim 1 wherein the target nucleic acid sequence
is characteristic of a human leukocyte antigen (HLA).
8. The method of claim 1 wherein the nucleotide sequence of said
amplification primer comprises (1) a priming region at its 3'-end
which is substantially complementary to a second target nucleic
acid sequence on the complement of said sample nucleic acid, and
(2) a probe region located 5' to said priming region which is
substantially complementary to a second reference nucleic acid
sequence which is 3' to the second target nucleic acid sequence on
the complement of said sample nucleic acid strand wherein when said
second reference nucleic acid sequence is contiguous with said
second target nucleic acid sequence on the sample nucleic acid
strand then the priming region and probe region on the
amplification primer are separated by a spacer region of nucleic
acid.
9. A diagnostic primer for detecting the presence of a target
nucleic acid sequence on a sample nucleic acid strand comprising
(1) a priming region at its 3'-end which is substantially
complementary to the target nucleic acid sequence, and (2) a probe
region located 5' to said priming region which is substantially
complementary to a reference nucleic acid sequence which is 3' to
the target nucleic acid sequence on the sample nucleic acid strand
wherein when said reference nucleic acid sequence is contiguous
with said target nucleic acid sequence on the sample nucleic acid
strand then the priming region and probe region on the diagnostic
probe are separated by a spacer region of nucleic acid.
10. The diagnostic primer of claim 9 wherein said priming region
and said probe region on the diagnostic primer are separated by a
spacer region of nucleic acid sequence which is not complementary
to the sequence of the sample nucleic acid strand between the
target and reference sequences
11. The diagnostic primer of claim 10 wherein said spacer is from 1
to 30 bases long.
12. The diagnostic primer of claim 10 wherein said spacer is from 8
to 30 bases long.
13. The diagnostic primer of claim 9 wherein the target nucleic
acid sequence is characteristic of a human leukocyte antigen
(HLA).
14. The diagnostic primer of claim 9 wherein the priming region and
probe regions are exactly complementary to the target and reference
nucleic acid sequences respectively.
15. A kit comprising a diagnostic primer for detecting the presence
of a target nucleic acid sequence on a sample nucleic acid strand
comprising (1) a priming region at its 3'-end which is
substantially complementary to the target nucleic acid sequence,
and (2) a probe region located 5' to said priming region which is
substantially complementary to a reference nucleic acid sequence
which is 3' to the target nucleic acid sequence on the sample
nucleic acid strand wherein when said reference nucleic acid
sequence is contiguous with said target nucleic acid sequence on
the sample nucleic acid strand then the priming region and probe
region on the diagnostic primer are separated by a spacer region of
nucleic acid; each of four different nucleoside triphosphates; and
an agent for polymerization of the nucleoside triphosphates.
16. The kit of claim 15 wherein said priming region and said probe
region on the diagnostic probe are separated by a spacer region of
nucleic acid sequence which is not complementary to the sequence of
the sample nucleic acid strand between the target and reference
sequences.
17. The kit of claim 15 further comprising an amplification primer
corresponding to said diagnostic primer, the nucleotide sequence of
the amplification primer being such that any extension product of
the corresponding diagnostic primer may, after separation from its
complement serve as a template for synthesis of an extension
product of the amplification primer.
18. The kit of claim 15 wherein the priming region and probe
regions of said diagnostic primer are exactly complementary to the
target and reference nucleic acid sequences respectively.
19. The kit of claim 15 wherein the nucleotide sequence of said
amplification primer comprises (1) a priming region at its 3'-end
which is substantially complementary to a target nucleic acid
sequence on the complement of said sample nucleic acid, and (2) a
probe region located 5' to said priming region which is
substantially complementary to a second reference nucleic acid
sequence which is 3' to the second target nucleic acid sequence on
the complement of said sample nucleic acid strand wherein when said
second reference nucleic acid sequence is contiguous with said
second target nucleic acid sequence on the sample nucleic acid
strand then the priming region and probe region on the diagnostic
primer are separated by a spacer region of nucleic acid.
20. The kit of claim 16 wherein said spacer is from 1 to 30 bases
long.
21. The kit of claim 16 wherein said spacer is from 8 to 30 bases
long.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to methods for the detection
of the presence or absence of nucleic acid sequences which are
characteristic of pathogens and the like as well as of gene
variations and mutations including those relating to the human
leukocyte antigen (HLA) which is of interest in the field of human
transplantation.
[0002] The HLA locus is highly polymorphic in nature. As disclosed
in the Nomenclature for Factors of the HLA System 1996 (Tissue
Antigens 1997: 49:297-321); there are 83 HLA-A alleles, 186 HLA-B
alleles, 42 HLA-C alleles, 184 HLA-DRB1 alleles, 11 DRB3 alleles, 8
DRB4 alleles, 12 DRB5 alleles, 18 DQA1 alleles and 31 DQB1 alleles,
with new alleles being discovered continuously. All HLA-A, -B, and
-C alleles have similar sequences. The same holds for DRB1, DRB3,
DRB4 and DRB5 sequences. Because of these similarities, very often
when a primer pair is used in the practice of polymerase chain
reaction sequence-specific priming (PCR-SSP), two or more alleles
will be amplified. Therefore, for each allele to have a unique
PCR-SSP pattern many pairs of primers must be used. Accordingly, in
clinical use of PCR-SSP for HLA typing there exists a desire to use
a limited number of PCR reactions to achieve as much resolution as
possible whereby the number of alleles amplified by a pair of
primers would be reduced (i.e., the specificity of the primers
increased). Simultaneously, all of the primer pairs must have
optimal annealing temperatures within a very restricted range.
[0003] PCR requires a pair of primers flanking the region on the
DNA template for that region to be amplified. The ability of a
primer to anneal to the desired sequence depends on the length of
the primer and the annealing temperature set in the PCR
thermocycling program. The longer the primer, the higher the
annealing temperature it needs to achieve specific amplification of
a DNA sequence. If the annealing temperature for a PCR is above the
optimal range for the primer to anneal to its target, little or no
amplification will occur. If the annealing temperature for a PCR is
below the optimal range for the primer to anneal to its target,
non-specific amplification will occur. PCR-SSP uses a balance
between primer length and annealing temperature to achieve the
specificity of the primer-directed sequence amplification. This
technique can be used to characterize the sequence on the target
DNA template--if amplification occurs, the template DNA contains
the same sequences as the primers used; if no amplification occurs,
the sequences on the template DNA are different from the primer
sequences. Of interest to the present application are the
disclosures of Olerup et al., Tissue Antigens 41 119-134 (1993) and
Bunce et al., Tissue Antigens 43: 7-17 (1994) which teach methods
of PCR-SSP for HLA typing.
[0004] Newton et al., U.S. Pat. No. 5,595,890 disclose PCR
diagnostic methods for typing including molecular typing of HLA
using PCR-SSP. According to this method, an unknown allele is
assigned based on the pattern of positive or negative reactions
from multiple PCR. The methods disclosed by Newton are limited in
their effectiveness for HLA typing, however, due to the high degree
of polymorphism in HLA as described above. As a consequence two
primers, each with specific sequences, frequently amplify many HLA
alleles, thus increasing the required number of PCR in order to
assign an unknown allele.
[0005] Accordingly, there exists a desire in the art for improved
methods of PCR-SSP based molecular typing whereby the specificity
of the typing can be increased so as to reduce the number of PCR
reactions required for each typing.
SUMMARY OF THE INVENTION
[0006] The present invention relates to improved methods for
detecting and/or amplifying target nucleic acid sequences and in
particular for the detection and amplification of human leukocyte
antigens (HLA) through methods such as polymerase chain reaction
whereby the specificity of diagnostic primers is increased such
that at least one primer is capable of recognizing two or more
regions on the template and is preferably capable of doing so
without increasing the annealing temperature of the primer to the
template DNA. The increased specificity of the primer set reduces
the number of alleles amplified by that primer pair and provides
improved resolution over conventional PCR-SSP at lower cost.
[0007] Specifically, the invention provides a method for detecting
the presence of a target nucleic acid sequence on a sample nucleic
acid strand from an individual comprising the steps of: treating
the sample, together or sequentially with appropriate nucleoside
triphosphates, an agent for polymerization of the nucleoside
triphosphates, a diagnostic primer and an amplification primer
under hybridizing conditions; wherein the nucleotide sequence of
said diagnostic primer comprises (1) a priming region at its 3'-end
which is substantially complementary to the target nucleic acid
sequence, and (2) a probe region located 5' to said priming region
which is substantially complementary to a reference nucleic acid
sequence which is 3' to the target nucleic acid sequence on the
sample nucleic acid strand wherein when said reference nucleic acid
sequence is contiguous with said target nucleic acid sequence on
the sample nucleic acid strand then the priming region and probe
region on the diagnostic probe are separated by a spacer region of
nucleic acid, whereby for selected hybridization and extension
conditions an extension product of the diagnostic primer is
synthesized when the priming region is substantially complementary
to the target nucleic acid sequence and when the probe region is
substantially complementary to the reference nucleic acid sequence,
but wherein for said selected hybridization and extension
conditions no extension product of the diagnostic primer is
synthesized when either the priming region or the probe region are
not substantially complementary to the target and reference nucleic
acid sequences respectively; any extension product of the
diagnostic primer formed being capable of serving as a template for
synthesis of an extension product of said amplification primer
after separation from its complement; amplifying any extension
product; and detecting the presence or absence of the target
polynucleic acid sequence from the presence or absence of
amplification product obtained as above. According to a preferred
aspect of the present invention, the priming region and probe
regions are exactly complementary to the target and reference
nucleic acid sequences respectively.
[0008] While the priming region and the probe region can be
contiguous on the diagnostic primers of the invention they need not
be. Thus, according to one preferred embodiment of the invention
the priming region and the probe region may be separated by a
spacer region of nucleic acid sequence which is not complementary
to the sequence of the sample nucleic acid strand between the
target and reference sequence. Specifically, the spacer region is
selected so as to provide a discontinuity in the complementarity of
the nucleotide sequences of the priming and probe regions of the
diagnostic primer such that hybridization and extension of the
primer region will not occur unless the probe region is
substantially complementary to the reference nucleic acid
sequence.
[0009] The spacer region can be from 1 to 30 nucleotides or more in
length with lengths of from 8 to 30 nucleotides being preferred
although those of skill in the art will recognize that in general
the longer the spacer region the longer the priming region must be
in order to successfully hybridize to the target nucleic acid
sequence under the hybridization conditions selected for practice
of the invention. Nevertheless, the length of the spacer region, as
well as the length of intervening sequence between the target
nucleic acid sequence and the reference nucleic acid sequence on
the sample nucleic acid strand which can be from 0 to 350 bases or
more should not be so long that the priming region is able to
hybridize independently to the target nucleic acid sequence by
virtue of increased length (and therefore increased annealing
temperature) without requiring prior hybridization of the probe
region to the reference nucleic acid sequence.
[0010] The present invention additionally provides kits for
practice of the methods of the invention which comprise a
diagnostic primer according to the invention in combination with
each of four nucleoside triphosphates and an agent for
polymerization of the nucleoside triphosphates. The kits can also
optionally comprise an amplification primer being such that any
extension product of the corresponding diagnostic primer may, after
separation from its complement, serve as a template for synthesis
of an extension product of the amplification primer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIGS. 1a, 1b and 1c depict a first embodiment of the present
invention wherein the reference sequence is 3' to the target
sequence on the sample nucleic acid strand; and
[0012] FIGS. 2a, 2b and 2c depict a second embodiment of the
present invention wherein the reference sequence is contiguous with
the target sequence on the sample nucleic acid strand and the
diagnostic primer comprises a spacer sequence disposed between the
probe region and the priming region.
DETAILED DESCRIPTION
[0013] The present invention relates to nucleic acid primers which
may be used in a variety of manners but which are particularly
useful for use in PCR amplification of DNA Rather than using a
conventional design of primer, which both recognizes a DNA sequence
by complementarity and acts as a primer for synthesis by a DNA
polymerase, the present invention provides primers characterized by
having two different functions which comprise two (or more)
non-consecutive DNA sequences on the same primer. According to the
invention, first portion of the primer sequence, the probe region,
functions primarily to specifically bind to a reference nucleic
acid sequence on the target nucleic acid which is 3' to and
preferably non-contiguous with the target nucleic acid sequence
without having the restriction of being exactly complementary at
the 3'-end, (which is usually required for efficient priming for a
DNA polymerase to produce an extension product). Coupled at the
3'-end of the probe region is the priming region, which recognizes
the target sequence on the sample nucleic acid strand. The priming
region is preferably separated downstream from the probe region but
may be contiguous if provided the reference nucleic acid sequence
and target nucleic acid sequence on the sample nucleic acid strand
are not contiguous. The priming region can also increase
specificity of the primer as a whole and must be sufficiently
complementary to the target sequence so that it can function to
promote extension of the target sequence under appropriate
hybridization and extension conditions.
[0014] According to practice of the invention the probe region
functions to roughly align the primer to the region of interest on
the sample nucleic acid strand (which according to one embodiment
of the invention is the HLA region). The priming region is thereby
more favorably disposed kinetically (by virtue of a local
concentration effect) such that it can thereafter more readily
recognize another nearby sequence (usually within a few hundred
base pairs). Increased specificity for a particular allele can be
provided if a suitable specific DNA sequence is nearby.
[0015] The priming sequence may add specificity to binding of the
diagnostic primer, but also functions as the site to prime DNA
synthesis by the DNA polymerase (usually a heat stable polymerase
such as Taq polymerase). By using spacings for the target sequences
that are within a restricted range, e.g. 50-350 bp on the template
DNA, amplification products for virtually any allele can be
separated from larger or smaller standard bands even on gel systems
designed for a restricted size range (such as that described in
U.S. Pat. No. 5,785,835 and available commercially as the Micro-SSP
gel box, One Lambda, Inc. Canoga Park, Calif.). Practice of the
present invention also provides particular advantages because
keeping the use of shorter amplification products avoids problems
associated with weak amplification of larger products in fast PCR
programs.
[0016] The diagnostic primers of the present invention are
particularly useful for use in carrying out the PCR reaction which
typically relies on the sequence specificity of two primers used in
the amplification step. Use of the diagnostic primers of the
invention allows the PCR reaction to utilize more than two regions
of sequence-specificity in a targeted DNA because each primer can
contain more than one sequence-specificity. According to practice
of the invention the length of the probe region can be shorter than
would otherwise be optimal under selected hybridization and
extension conditions as long as the probe region will recognize the
target sequence with moderate efficiency. After the probe region
anneals to the target sequence, the priming region will then
readily anneal to its downstream target sequence.
[0017] The length of priming region should be selected to be below
the length that would otherwise be optimal under the selected
hybridization and extension conditions otherwise it would anneal to
the target sequence on its own (and allow an extension product to
be formed). Annealing of the probe region to the reference nucleic
acid sequence on a nucleic acid strand containing the target
sequence will increase the local concentration of the priming
sequence to its target sequence, thus achieving successful
annealing of the priming region to its target under sub-optimal
conditions. Therefore, the priming region and the probe region are
designed such that under given selected hybridization and extension
conditions, an extension product of the diagnostic primer is
synthesized when the priming region is substantially complementary
to the target nucleic acid sequence and when the probe region is
substantially complementary to the reference nucleic acid sequence,
but wherein no extension product of the diagnostic primer is
synthesized under those same hybridization and extension conditions
when either the priming region or the probe region are not
substantially complementary to the target and reference nucleic
acid sequences respectively. Nevertheless, it will be understood
that the selectivity of the diagnostic primers of the invention is
dependent upon the hybridization and extension conditions selected
for practice. Thus, those of skill in the art will recognize that
higher annealing temperatures will generally require longer primer
and probe sequences while lower annealing temperatures will
generally require shorter primer and probe sequences. Other factors
such as reagent concentrations and the GC content of the target and
reference regions of the sample nucleic acid strand will also
affect the design of the diagnostic primers of the invention.
[0018] FIG. 1 depicts an embodiment of the present invention
whereby a diagnostic primer comprising a probe region and a priming
region which are not separated by a spacer region is used in
combination with an amplification primer to detect the presence of
a target nucleic acid sequence on a sample nucleic acid strand.
Specifically, FIG. 1a depicts the hybridization of the probe region
to a reference nucleic acid sequence which is 3' to the target
nucleic acid sequence on the sample nucleic acid. Hybridization of
the probe sequence to the reference sequence creates a local
concentration effect such that under the selected hybridization and
extension conditions the priming region of the diagnostic primer
will hybridize to the target sequence on the sample nucleic. The
hybridization and extension conditions are further selected given
the identity of the target and the diagnostic primer that the
priming region will not hybridize with the target sequence when the
probe region is not hybridized to the associated reference
sequence. FIG. 1a further depicts hybridization of an amplification
primer to another sequence associated with the target nucleic acid
sequence. FIG. 1b depicts hybridization of the probe region and
priming region of the diagnostic primer to the reference sequence
and target sequence on the sample nucleic acid respectively and the
formation of a "hairpin" by the intervening sequence between the
reference sequence and the target sequence on the sample nucleic
strand which have hybridized to portions of the diagnostic primer.
FIG. 1c depicts extension of the diagnostic and amplification
primers under extension conditions in the presence of appropriate
nucleoside triphosphates and a polymerase. The extension product of
the diagnostic primer may then be used as a template for synthesis
of an extension product of the amplification primer after
separation from its complement and the extension product may be
amplified and detected according to conventional methods.
[0019] FIG. 2 depicts a second embodiment of the present invention
whereby a diagnostic primer comprising a probe region and a priming
region separated by a spacer region is used in combination with an
amplification primer to detect the presence of a target nucleic
acid sequence on a sample nucleic acid strand. In this case, the
target nucleic acid sequence is associated with a reference nucleic
acid sequence which is contiguous with the target nucleic acid
sequence on the sample nucleic acid strand. Specifically, FIG. 2a
depicts the hybridization of the probe region to a reference
nucleic acid sequence which is 3' to the target nucleic acid
sequence on the sample nucleic acid. Hybridization of the probe
sequence to the reference sequence creates a local concentration
effect such that under the selected hybridization and extension
conditions the priming region of the diagnostic primer will
hybridize to the target sequence on the sample nucleic. The
hybridization and extension conditions are further selected given
the identity of the target and the diagnostic primer that the
priming region will not hybridize with the target sequence when the
probe region is not hybridized to the associated reference
sequence. FIG. 2a further depicts hybridization of an amplification
primer to another sequence associated with the target nucleic acid
sequence. FIG. 2b depicts hybridization of the probe region and
priming region of the diagnostic primer to the reference sequence
and target sequence on the sample nucleic acid respectively and the
formation of a "hairpin" by the spacer region between the probe
region and the priming region which is not complementary to the
sequence of the sample nucleic acid strand between the target and
reference sequences. FIG. 2c depicts extension of the diagnostic
and amplification primers under extension conditions in the
presence of appropriate nucleoside triphosphates and a polymerase.
The extension product of the diagnostic primer may then be used as
a template for synthesis of an extension product of the
amplification primer after separation from its complement and the
extension product may be amplified and detected according to
conventional methods.
[0020] The present invention provides improvements over prior art
typing methods such as those of Newton et al., U.S. Pat. No.
5,595,890 where multiple PCR reactions may be required to assign an
unknown allele. Use of the diagnostic primers of the invention will
add extra selectivity to the sequence specific primer method
because more than two unique sequences can be used as the selection
criteria for the PCR. Thus, the number of separate PCR reactions
required for assigning an unknown allele may be reduced which
reduces the cost of PCR-SSP testing. Selection of appropriate
primers according to the invention will allow resolution of
ambiguities that occur in some heterozygous cases wherein the
multi-PCR pattern derived from two different alleles is identical
to another pair of alleles.
[0021] The use of primers according to the invention allows greater
specificity in the recognition of a specific allele or set of
alleles by using more than one region of sequence homology to the
nucleic acid sequence of interest. Increasing the specific
recognition of nucleic acid sequence homology refines the ability
to carry out a variety of DNA-based tests. Included among these
tests would be HLA tissue typing, detection of genetically
inherited diseases, detection of infectious organisms in tissue, or
detection of a variety of other markers or conditions based on the
presence of a nucleic acid sequence (e.g. for testing the efficacy
of a gene therapy technique). The diagnostic primers of the
invention may also be used in methods for the transcriptional base
amplification and Q-Replicase base amplification.
[0022] It will be further appreciated that any extension product
obtained may if desired be amplified by the polymerase chain
reaction (PCR) as described in U.S. Pat. Nos. 4,683,195 and
4,683,202, by the use of Q-beta replicase described in PCT Patent
Publication WO 87/06270 and in Biotechnology Vol. 6, October 1988,
by the use of the transcription based nucleic acid amplification of
Siska Corporation as described in PCT Patent Publication WO
88/10315, or by the use of linear amplification. In this
connection, the expression "linear amplification" is used herein to
refer to amplification using a single primer for each diagnostic
portion in the presence of an agent for polymerization and
appropriate nucleotide triphosphates whereby amplification is
effected by primer extension based on the use of a single strand of
sample nucleic acid as template.
[0023] Where amplification is effected either by the use of
diagnostic and amplification primers or by the use of two
diagnostic primers, for example, as described in the first and
second embodiments of the present invention or as part of the
amplification procedure described in European Patent Publication
No. 237,362, the steps of (a) denaturing to separate primer
extension products from their template and (b) contacting single
strands thereby obtained, either together or sequentially, with
appropriate nuceloside triphosphates, an agent for polymerization
of the nucleoside triphosphates, and the relevant primers to
synthesize further extension produces; are preferably repeated at
least five times (cycles) up to an indefinite number, especially
where the sample DNA is refractory to amplification, without
detriment to the present invention. More preferably 15-60, e.g.,
15-30 times (cycles) are employed if the sample contains human
genomic DNA. If the sample comprises cells, preferably they are
heated to expose the nucleic acids therein to the reagents. This
step avoids purification of the nucleic acids prior to reagent
addition. In this regard, it will be appreciated that the present
invention represents a substantial improvement over prior processes
even if DNA purification from a sample is performed prior to the
attempted amplification.
[0024] It will be appreciated that contact between the single
strands produced by denaturation and the appropriate nucleoside
triphosphates, an agent for polymerization of the nucleoside
triphosphates, the primer(s), for example, the diagnostic primer(s)
and/or the amplification primer(s) may be effected either by
addition of these materials to the reaction mixture following
separation of the primer extension product from its template (step
a) or reliance may be placed on the materials already present in
the reaction mixture. Indeed, any one or more different nucleoside
triphosphates and/or the agent for polymerization and/or the
primer(s), for example, the diagnostic primer(s) and/or the
amplification primer may be added at any stage of the process of
the invention.
[0025] Linear amplification may be effected by any convenient means
and thus may be effected by the use of complementary nucleoside
triphosphates in the presence of an agent for polymerization of the
nucleoside triphosphates to produce primer extension products of
indeterminate length where a sufficient degree of complementarity
is present between the diagnostic primer and the sample nucleic
acid. Preferably, where all complementary nucleoside triphosphates
are to be employed, the sample nucleic acid is subjected to
endonuclease digestion, the restriction endonuclease being selected
so as to ensure that cleavage of the sample nucleic acid is
effected at a site adequate to permit the formation of primer
extension products of fixed length. Advantageously, however, the
linear amplification may be effected in the presence of only 1,
advantageously only 2 or preferably one 3 nucleoside triphosphates
such that the diagnostic primer in its bound state (i.e.,
hybridized to the sample nucleic acid) can only extend as far as
the presence of only the 1, 2 or 3 nucleoside triphosphates will
permit. Once a nucleoside triphosphate is present in the sample
nucleic acid for which no complementary nucleoside triphosphate is
present, then primer extension will cease.
[0026] If desired, the linear amplification may be effected at the
melting temperature (Tm) of the sequence. At this temperature, the
diagnostic primer hybridized to the complementary sequence in the
sample nucleic acid is in equilibrium with the diagnostic primer
free in solution and thus the diagnostic primer (optionally in
extended form) is being rapidly hybridized to and denatured from
the sample nucleic acid. If desired, the linear amplification may
also be effected by thermal oscillation. Such thermal oscillation
would generally involve rapid temperature fluctuation about the
melting temperature of the sequence.
[0027] The term "nucleoside triphosphate" is used herein to refer
to nucleosides present in either DNA or RNA and thus includes
nucleosides which incorporate adenine, cytosine, guanine, thymine
and uracil as base, the sugar moiety being deoxyribose or ribose.
In general, deoxyribonucleosides will be employed in combination
with a DNA polymerase. It will be appreciated, however, that other
modified bases capable of base pairing with one of the conventional
bases adenine, cytosine, guanine, thymine and uracil may be
employed. Such modified bases include, for example, 8-azaguanine
and hypoxanthine.
[0028] The term "nucleotide" as used herein can refer to
nucleotides present in either DNA or RNA and thus includes
nucleotides which incorporate adenine, cytosine, guanine, thymine
and uracil as base, the sugar moiety being deoxyribose or ribose.
It will be appreciated, however, that other modified bases capable
of base pairing with one of the conventional bases, adenine,
cytosine, guanine, thymine and uracil, may be used in the
diagnostic primer and amplification primer employed in the present
invention. Such modified bases include, for example, 8-azaguanine
and hypoxanthine.
[0029] The agent for polymerization from the nucleoside
triphosphates 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 thermostable enzymes.
The term "thermostable enzyme" as used herein refers to any enzyme
which is stable to heat and is heat resistant and catalyzes
(facilitates) 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 will proceed in the 5'
direction along the template strand, until synthesis terminates,
producing molecules of different lengths. There may be enzymes for
example, thermostable enzymes, however, which initiate synthesis at
the 5' end and proceed in the other direction, using the same
process as described above. A preferred thermostable enzyme which
may be employed in the process of the present invention is that
which can be extracted and purified from Thermus aquaticus. Such
enzyme has a molecular weight of about 86,000-90,000 daltons as
described in European Patent Publication No. 237,362 (see also
European Patent Publication No. 258,017). Thermus aquaticus strain
YT1 is available without restriction from the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md., USA as ATCC
25,104.
[0030] The term "complementary to" is used herein in relation to
nucleotides to mean a nucleotide which will base pair with another
specific nucleotide. Thus, adenosine triphosphate is complementary
to uridine triphosphate or thymidine triphosphate and guanosine
triphosphate is complementary to cytidine triphosphate. It is
appreciated that whilst thymidine triphosphate and guanosine
triphosphate may base pair under certain circumstances, they are
not regarded as complementary for the purpose of this
specification. It will also be appreciated that whilst cytosine
triphosphates and adenosine triphosphate may base pair under
certain circumstances, they are not regarded as complementary for
the purposes of this specification. The same applies to cytosine
triphosphate and uracil triphosphate.
[0031] 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. Thus, primer sequences (including the probe or
priming regions in the diagnostic primer) do not necessarily have
to be exactly complementary to the target sequences. Generally a
small number of mismatches will be tolerated in the middle of the
primer sequences and will allow hybridization and priming of
extension products. In general, the degree of mismatching tolerated
depends upon the primer length which in turn affects the
denaturation temperature and the annealing temperature selected for
practice of the PCR cycles. If the denaturation temperature of the
primer is close to or higher than the annealing temperature (less
stringent), then the primer will still prime to the target sequence
despite a small number of (generally one or two or at most three)
mismatches. The probe region may be capable of tolerating more
mismatches in the middle of the sequence but it still depends on
the denaturation temperature of the probe region and the annealing
temperature of the selected PCR hybridization and extension
conditions. Nevertheless, it is preferred that the priming and
probe sequences be exactly complementary to their respective
targets.
[0032] The term "amplification primer" is used herein to refer to a
primer which is capable of hybridizing to the nucleic acid strand
which is complementary to the nucleic acid strand to which the
diagnostic primer is capable of hybridizing, the "amplification
primer" having a nucleotide sequence such that it is capable of
hybridizing to a diagnostic primer extension product, after
separation from its complement, whereby the diagnostic primer
extension product serves as a template for synthesis of an
extension product of the amplification primer, thereby facilitating
amplification. It should further be recognized that a second
diagnostic primer of the invention which is specific for a second
target sequence and second reference sequence may also be used as
the amplification primer such that the nucleotide sequence of the
amplification primer comprises (1) a priming region at its 3'-end
which is substantially complementary to a target nucleic acid
sequence on the complement of said sample nucleic acid, and (2) a
probe region located 5' to said priming region which is
substantially complementary to a second reference nucleic acid
sequence which is noncontiguous with and 3' to the second target
nucleic acid sequence on the complement of said sample nucleic acid
strand.
[0033] A much simpler and preferred method of distinguishing
between amplification products comprises selecting the nucleotide
sequences of the amplification primers such that the length of each
amplified product formed during the process of the present
invention is different. In this regard the number of base pairs
present in an amplification product is dictated by the distance
between the target sequences of the diagnostic and amplification
primers. Thus, the amplification primers may be designed such that
each potential variant nucleotide is associated with a potential
amplification product of different length.
[0034] The presence or absence of a given potential variant
nucleotide may thus advantageously be detected by electrophoretic
techniques, in which the different amplified products obtained may
be distributed according to their molecular weight and thereby
identified for example by autoradiography or fluorescent
techniques.
[0035] Other aspects and advantages of the present invention will
be understood upon consideration of the following illustrative
examples.
EXAMPLE 1
[0036] According to this example, a single diagnostic primer
according to the invention comprising a priming region and a probe
region was used to distinguish the following HLA alleles
(DRB1*03021, *03022, *0303 (serologically defined as DR18)) from
the rest of the DRB1*03 alleles which are generally defined
serologically as DR17. The target DNA was isolated from
EBV-transformed human B-cell line using QIAmp Blood Kit (Qiagen,
Inc. Santa Clara, Calif.).
[0037] As a control, primer OLR-14(5'-CTTGGAGTACTCTACGTCT-3'; 10
pmole) (SEQ ID NO: 1) co-responding to amino acid 8-13 of DRB1 and
primer OLR-142(5'-GCAGTAGTTGTCCACCCG-3'; 10 pmole) (SEQ ID NO: 2)
corresponding to amino acid 74-79 were used as PCR primers
according to conventional practice wherein all PCR reactions are
performed using a Perkin-Elmer Gene-Amp PCR System 9600the
following conditions: 10 mM Tris(hydroxymethyl)aminomethane
Hydrochloride (pH 8.3@25(C); 50 mM Potassium Chloride, 14.5 mM
Magnesium Chloride, 0.001% (w/v) Gelatin, 200(M dNTP's (dATP, dCTP,
dGTP, dTTP), 6% (w/v) Sucrose, 0.002% (w/v) Cresol Red at 10
microliters total reaction volume with the PCR Reaction profile set
out below:
1 No. of Cycles Step Temp (.degree. C.) Time (sec.) 1 1 96 130 2 63
60 9 1 96 10 2 63 60 20 1 96 10 2 59 50 3 72 30
[0038] Primers OLR-147 and OLR-148 which amplify a 750 base-pair
fragment of the Human beta-Globin gene were included in all PCR to
serve as the internal control for proper PCR amplification.
[0039] According to the control experiment, the following 13
(thirteen) DRB1* alleles were amplified: DRB1*03011, DRB1*03012,
DRB1*03021, DRB1*03022, DRB1*0303, DRB1*0304, DRB1*0305, DRB1*0306,
DRB1*0307, DRB1*0308, DRB1*0309, DRB1*03010, and DRB1*1107.
[0040] The experiment was then repeated with a primer according to
the invention containing a priming region and a probe region
separated by a spacer. Note: An extra "A" was inserted between the
probe and primer regions to separate these two sequences, because
the nucleotide at the 5'-end of the primer region, a "G", is the
same as the next nucleotide 3'-end to the probe region. Without
insertion of the "A" spacer, the probe region extends one more
nucleotide thus increasing the melting temperature of the probe
region by approximately 4.degree. C. Specifically, when diagnostic
primer OLR-1414 (5'-TTGGAGTACTCTACGTCTGAAGG- TTCCTGGAG-3'; 10
pmole) (SEQ ID NO: 3) co-responding to amino acids 8-14 (with
underlined sequence) and 25-28 (with bolded sequence) of DRB1 with
a gap of 32 base pairs and conventional amplification primer
OLR-142(5'-GCAGTAGTTGTCCACCCG-3'; 10 pmole) (SEQ ID NO:2)
co-responding to amino acid 74-79 were used, only the following
three alleles were amplified: DRB1*03021, DRB1*03022, and
DRB1*0303. Accordingly, use of the Primer OLR-1414 provides a
substantial improvement over the use of conventional primers.
EXAMPLE 2
[0041] According to this example, the method of the invention is
practiced in a manner which demonstrates that because the primer
region of the diagnostic primers of the invention can direct the
PCR reaction under the sub-optimal condition, that a mismatch in
the primers is not required to be at the 3'-end of the primer to
perform successful PCR-SSP. Specifically, the primers OLR-1419
(5'-TTGGAGTACTCTACGTCTGATTCCTGGAGAGA-3- '; 10 pmole) (SEQ ID NO: 4)
and OLR-1420(5'-TTGGAGTACTCTACGTCTGATCCTGGAGAG- ATA-3'; 10 pmole)
(SEQ ID NO:5) which have the same probe region(the underlined
sequence) but have slightly different primer regions to OLR-1414
(5'-TTGGAGTACTCTACGTCTGAA-GGTTCCTGGAG-3'; 10 pmole) (SEQ ID NO: 3)
were used to amplify target DNA according to the method of Example
1. The differences between DRB1*0301 and DRB1*03021, *03022, *0303
are indicated by bold and underlined residues. When these three
primers are individually paired with OLR-142 as an amplification
primer, all primer pairs successfully amplify the DRB1*03021 allele
but not the DRB1*0301 allele. This example demonstrates that even
with the mismatch between DRB1*0301 and DRB1*03021, *03022, *0303 6
nucleotides away from the 3'-end, the primer of the invention can
still be used to selectively amplify the desired allele.
EXAMPLE 3
[0042] According to this example two primers comprising priming
regions and probe regions separated by a spacer region were used to
amplify target sequences according to the methods of the invention.
Specifically, OLR-1334 (5'-GACCGGAACACACGGTTGGCCCAGT-3') (SEQ ID
NO: 6) which recognizes the amino acid sequence of 61-65 and 69-71
of the HLA-A (mature protein sequence) and
OLR-1348(5'-ACTCACCGTCCTCGCAATAGTAGCC-3') (SEQ ID NO: 7) which
recognizes the amino acid sequence of 91-88(plus 6 nucleotides of
the intron 3) and 85-83 of the HLA-A were used to perform PCR. The
underlined sequence in these two primers represents the probe
region and the bolded sequence represents the primer region. A "TT"
sequence was inserted between the probe and primer region as a
spacer. This primer pair amplifies only the following alleles:
A*3401, A*3402, and A*6601.
[0043] However, if only the probe region of each primer were used
as the PCR-SSP primers, the following extra alleles will be
amplified: A-2501, A-2502 A-2601, A-2602, A-2603, A-2604, A-2605,
A-2606, A-2608, A-2609, A-2610, and A-2611N. Accordingly, the
primers of the present invention can be effectively used as pairs
of primers in polymerase chain reaction to increase the specificity
of PCR-SSP.
[0044] Numerous modifications and variations in the practice of the
invention are expected to occur to those skilled in the art upon
consideration of the presently preferred embodiments thereof.
Sequence CWU 1
1
7 1 19 DNA Artificial Sequence Description of Artificial Sequence
primer OLR-14 1 cttggagtac tctacgtct 19 2 18 DNA Artificial
Sequence Description of Artificial Sequence primer OLR-142 2
gcagtagttg tccacccg 18 3 32 DNA Artificial Sequence Description of
Artificial Sequence primer OLR-1414 3 ttggagtact ctacgtctga
aggttcctgg ag 32 4 32 DNA Artificial Sequence Description of
Artificial Sequence primer OLR-1419 4 ttggagtact ctacgtctga
ttcctggaga ga 32 5 33 DNA Artificial Sequence Description of
Artificial Sequence primer OLR-1420 5 ttggagtact ctacgtctga
tcctggagag ata 33 6 25 DNA Artificial Sequence Description of
Artificial Sequence primer OLR-1334 6 gaccggaaca cacggttggc ccagt
25 7 25 DNA Artificial Sequence Description of Artificial Sequence
primer OLR-1348 7 actcaccgtc ctcgcaatag tagcc 25
* * * * *