U.S. patent application number 12/398341 was filed with the patent office on 2010-11-18 for compositions and methods for determining genotypes.
This patent application is currently assigned to Quest Diagnostics Investments Incorporated. Invention is credited to Kevin Z. Qu, Anthony Sferruzza.
Application Number | 20100291554 12/398341 |
Document ID | / |
Family ID | 32717603 |
Filed Date | 2010-11-18 |
United States Patent
Application |
20100291554 |
Kind Code |
A1 |
Qu; Kevin Z. ; et
al. |
November 18, 2010 |
COMPOSITIONS AND METHODS FOR DETERMINING GENOTYPES
Abstract
The present invention provides methods for determining the
genotype of a selected gene present in at least two alleles in a
sample. The methods involve amplifying DNA from the sample with a
first pair of flanking primers that hybridize to nucleic acid
sequences flanking a variant-specific gene sequence, the presence
of which indicates the presence of a first gene variant, and the
absence of which indicates the presence of a second gene variant.
The DNA is also amplified with a third primer that specifically
binds to the variant-specific sequence and together with one of the
flanking primers forms a second pair of primers. Detection of one
or more nucleic acid products of the amplification reaction is
indicative of the genotype present in the sample.
Inventors: |
Qu; Kevin Z.; (Irvine,
CA) ; Sferruzza; Anthony; (San Clemente, CA) |
Correspondence
Address: |
FOLEY & LARDNER LLP
P.O. BOX 80278
SAN DIEGO
CA
92138-0278
US
|
Assignee: |
Quest Diagnostics Investments
Incorporated
|
Family ID: |
32717603 |
Appl. No.: |
12/398341 |
Filed: |
March 5, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10714508 |
Nov 14, 2003 |
7514213 |
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12398341 |
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60426639 |
Nov 15, 2002 |
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Current U.S.
Class: |
435/6.11 ;
435/6.14 |
Current CPC
Class: |
C12Q 2600/156 20130101;
C12Q 1/6883 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of determining the presence or absence of an insertion
in a gene of interest in a sample obtained from a subject,
comprising: amplifying DNA in a single amplification reaction from
the sample with a first pair of flanking primers that hybridize to
nucleic acid sequences flanking an insertion sequence, and a third
primer that specifically binds to said insertion sequence and
together with one of the flanking primers forms a second pair of
primers; and identifying a homozygous genotype lacking said
insertion by detecting the production of one amplification product;
identifying a homozygous genotype having said insertion by
detecting the production of two amplification products, and
identifying a heterozygous genotype by detecting the production of
three amplification products.
2. The method of claim 1, wherein said amplification reaction is by
polymerase chain reaction.
3. The method of claim 1, wherein the sample is a human sample.
4. The method of claim 1, wherein the DNA is un-degraded DNA.
5. The method of claim 3, wherein the sample is a tissue
sample.
6. The method of claim 3, wherein the sample is selected from the
group consisting of blood, cultured cells, cells derived from
amniotic fluid, and cells derived from chorionic villi.
7. The method of claim 3 wherein the sample is blood.
8. The method of claim 3, wherein the DNA sample is from a source
selected from the group consisting of: the endothelium of blood
vessels, epithelial cells, blood mononuclear cells, macrophages,
male germinal cells, and a biological fluid.
9. The method of claim 1, wherein the gene of interest is selected
from the group consisting of genes encoding cytochrome P450
enzymes, insulin receptors, neurofibromatosis type 1, plaktoglobin,
and dipeptidyl carboxypeptidase-1.
10. The method of claim 1, wherein said insertion is a single
nucleotide polymorphism or a multiple nucleotide polymorphism.
11. The method of claim 1, wherein said DNA is cDNA.
12. The method of claim 1, wherein one of said first pair of
flanking primers comprises a detectable label.
13. The method of claim 12, wherein said detectable label is a
fluorescent label.
14. The method of claim 1, wherein said third primer comprises a
detectable label.
15. The method of claim 14, wherein said detectable label is a
fluorescent label.
16. The method of claim 1, wherein said sample is used to identify
a subject at risk for a disease selected from the group consisting
of essential hypertension, diabetic neuropathy, renal disease,
congestive cardiomyopathies and myocardial infarction.
17. The method of claim 1, wherein said sample is used to identify
in said subject an altered responsiveness to pharmaceutical
intervention.
18. The method of claim 17, wherein said pharmaceutical
intervention is selected from the group consisting of an ACE
inhibitor and an angiotensin II type 1 receptor antagonist.
19. The method of claim 18, wherein said ACE inhibitor is selected
from the group consisting of benazepril, captopril, cilazapril,
enalapril, enalaprilat, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, and trandolapril.
20. The method of claim 18, wherein said angiotensin II type 1
receptor antagonist is selected from the group consisting of
irbesartan, losartan, valsartan, telmisartan, camdesartam, and
eprosartan.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the identification of the
genotype of a subject, and the use of such identification for
diagnostic, prognostic, and therapeutic purposes.
BACKGROUND OF THE INVENTION
[0002] The following description of the background of the invention
is provided simply as an aid in understanding the invention and is
not admitted to describe or constitute prior art to the
invention.
[0003] Angiotensin converting enzyme (ACE) is a zinc
metalloproteinase involved in the renin-angiotensin and in the
kallikrein-kinin systems, in which it is responsible for the
proteolytic activation of angiotensin I and bradykinin. Because of
the central role played by the renin-angiotensin and
kallikrein-kinin systems in regulating blood pressure and
electrolyte balance, ACE has been identified as an important
therapeutic target for diseases such as essential hypertension,
diabetic neuropathy, renal disease, congestive cardiomyopathies
including congestive heart failure, and myocardial infarction. See,
e.g., Cambien et al., Nature 359: 641-44 (1992); U.S. Pat. No.
5,359,045; Higaki et al., Circulation 101: 2060-65 (2000); Kennon
et al., Diabet. Med. 16: 448-58 (1999). ACE has also been
identified as a risk factor for stent restenosis following
treatment for coronary artery disease. See, e.g., Ribichini et al.,
Circulation 97: 147-54 (1998).
[0004] ACE is mainly located on the endothelium of blood vessels,
especially in the pulmonary circulation, but it is also found in
epithelial cells, in blood mononuclear cells, in macrophages, in
male germinal cells and in a circulating form in several biological
fluids. Circulating ACE probably originates from the vascular
endothelial cells. In plasma and on the surface of endothelial
cells, ACE converts the inactive decapeptide angiotensin I into the
highly vasoactive and aldosterone-stimulating octapeptide
angiotensin II. Angiotensin II is a powerful vasoconstrictor which
may modulate or induce the growth of vascular smooth muscle cells
and cardiomyocytes. ACE can affect the oxidation of low density
lipoproteins (LDLs), endothelial cell function, and smooth muscle
cell migration and proliferation, which are all important
components of atherosclerosis.
[0005] The human ACE gene is located on chromosome 17q23 and
includes 26 exons. Its coding sequence is 4.3 kb in length and
codes for a protein of 1,306 amino acids. The ACE gene is present
in the population as different allelic variants. A variant of
particular interest clinically is the presence or absence of a 287
base pair ("bp") non-coding fragment within Intron 16. When this
287 by sequence is present in an ACE gene, the genotype is
designated "I" for "insertion"; conversely, when this 287 by
sequence is absent in an ACE gene, the genotype is designated "D"
for "deletion." Because the genome contains two copies of each
gene, referred to as "alleles," possible ACE genotypes with regard
to this variant are D/D, I/D, and I/I.
[0006] Increased ACE activity correlates strongly with the
deletion/deletion (D/D) and insertion/deletion (I/D) genotypes. The
D/D genotype has also been associated with myocardial infarction,
ischemic and idiopathic dilated cardiomyopathy, sudden death in
hypertrophic cardiomyopathy, and restenosis after percutaneous
transluminal coronary angioplasty. In addition, an increased risk
of coronary artery disease is attributed to the ACE D/D genotype.
The ACE genotype of an individual has also been related to response
to ACE inhibitors (such as benazepril, captopril, cilazapril,
enalapril, enalaprilat, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, and trandolapril) and to
angiotensin II type 1 receptor antagonists (such as irbesartan,
losartan, valsartan, telmisartan, camdesartam, and eprosartan).
See, e.g., Kurland et al., J. Hypertens. 19: 1783-87 (2001);
Okumura et al., Circ. J. 66: 311-16 (2002).
[0007] Polymerase chain reaction ("PCR") amplification, followed by
agarose gel electrophoresis, is commonly used to identify the ACE
genotype present in a sample. It has been reported, however, that
such PCR methods result in significant mistyping. See, e.g.,
Odawara et al., Hum. Genet. 100: 163-66 (1997); Shanmugan et al.,
PCR Methods Applications 3: 120-21 (1993); Rigat et al., Nucl. Acid
Res. 20: 1433 (1992). To eliminate the mistyping, a second PCR
reaction that detects only the I/I and I/D genotypes is typically
performed to confirm in the D/D genotype. Since only the I/I and
I/D genotypes can be detected in the second reaction, the absence
of a PCR fragment is taken as indicating a true D/D genotype. Such
methods, however, cannot distinguish an unsuccessful PCR reaction
from a true D/D genotype.
[0008] Each publication and patent in the foregoing section is
hereby incorporated by reference in its entirety, including all
tables, figures, and claims.
SUMMARY OF THE INVENTION
[0009] The present invention provides methods and compositions for
determining the genotype of a selected gene present in a sample.
While described hereinafter in reference to the angiotensin
converting enzyme (ACE) genotype present in a sample, the skilled
artisan will readily understand that the methods described herein
are generally applicable to the analysis of genes that are present
in one or more allelic variants.
[0010] The methods described herein comprise amplifying DNA from
the sample with a first pair of primers that hybridize to nucleic
acid sequences flanking a variant-specific gene sequence; that is,
a sequence, the presence of which indicates the presence of a first
gene variant, and the absence of which indicates the presence of a
second gene variant. A third primer is also provided that
specifically binds to the variant-specific sequence and together
with one of the flanking primers forms a second pair of primers.
One or more nucleic acid products of the amplification are
detected, and the nucleic acid products indicate the genotype
present in the sample. In one embodiment, three nucleic acid
products of the amplification are detected.
[0011] In preferred embodiments, the methods described herein
comprise amplifying DNA from the sample with a first pair of
primers that hybridize to nucleic acid sequences flanking a
variant-specific ACE sequence, the presence of which indicates the
presence of a first ACE gene variant, and the absence of which
indicates the presence of a second ACE gene variant. A third primer
is also provided that specifically binds to the variant-specific
ACE sequence and together with one of the flanking primers forms a
second pair of primers. One or more nucleic acid products of the
amplification are detected, and the nucleic acid products indicate
the ACE genotype present in the sample. In one embodiment three
nucleic acid products of the amplification are detected.
[0012] In addition to the ACE gene, the present invention may be
adapted to determine the gene variants present in any gene of
interest. Preferably, the gene variants comprise the presence or
absence of a particular sequence. For example, insertion/deletion
variants are known to those of skill in the art in genes encoding
cytochrome P450 enzymes (e.g., van der Weide and Steijns, Ann.
Clin, Biochem. 36: 722-29 (1999)); insulin receptors (e.g., Zee et
al., J. Hypertens, Suppl. 12: S13-22 (1994)); neurofibromatosis
type 1 (e.g., Grifa et al., Clin. Genet. 47: 281-84 (1995));
plaktoglobin (e.g., Protonotarios et al., J. Am. Coll. Cardiol. 38:
1477-84 (2001)); dipeptidyl carboxypeptidase-1 (e.g., Morris and
Zee, Clin. Exp. Pharmacol., Physiol. 21: 919-24 (1994)); etc.
[0013] In various preferred embodiments, the methods involve
contacting a sample of DNA from a subject, most preferably a human,
with primers selected to amplify a region of the ACE gene
containing a sequence that is indicative of the ACE variant
present. The primers consist of two forward primers and one reverse
primer (or, alternatively, two reverse primers and one forward
primer). Each of the forward (reverse) primers form a primer pair
with the reverse (forward) primer. The first primer pair flanks the
sequence that is indicative of the ACE variant (e.g., a sequence
that may be inserted in the ACE gene in certain genotypes); thus,
amplification by this primer pair provides an amplicon, regardless
of the presence or absence of the variant sequence (e.g., the
potential insertion). The second primer pair contains one primer
within the sequence that is indicative of the ACE variant (e.g.,
the potential inserted sequence); thus, amplification by this
primer pair will only occur if the ACE variant is present. In one
embodiment the sample is un-degraded DNA. By "un-degraded DNA" is
meant a sample of DNA having a population of DNA similar to that
derived from live tissue. Live tissue contains un-degraded DNA, as
do samples of DNA derived from live tissue or tissue that has died
very recently. Persons of ordinary skill know that when cells die
the DNA begins to degrade and forms different gel patterns (e.g.,
in a polyacrylamide or other size-differentiating gel) than samples
of DNA derived from live tissue, due to the fragmentation of DNA
strands in the sample that form as DNA begins to degrade. Thus, in
various embodiments un-degraded DNA will contain at least 70% or at
least 80% or at least 90% of the DNA strands found in a
corresponding sample from live tissue, which contains un-degraded
DNA. In various embodiments the live tissue can be, for example,
blood, cultured cells, cells derived from amniotic fluid, or cells
derived from chorionic villi.
[0014] The present invention is described below in terms of the 287
base pair ("bp") non-coding fragment within Intron 16 that is
referred to by the artisan as the I/D polymorphism. A polymerase
chain reaction amplification of the sample of DNA is performed with
replication of the DNA being initiated by the three primers
described above. In this preferred embodiment, the size of the
first amplicon is indicative of the presence or absence of the
inserted sequence, and hence whether the D or I variant is present.
The presence of the second amplicon verifies the presence of the I
variant, while the absence of the second amplicon verifies the
presence of the D variant. Thus, the nucleic acid products of the
PCR amplification are detected, and the genotype is determined
based on the nucleic acid products of the PCR amplification:
##STR00001##
[0015] By "variant-specific ACE sequence" is meant a nucleic acid
sequence that, when present or absent, correlates to a particular
ACE variant present in a particular genotype. For example, in the
most preferred embodiment the variant-specific ACE sequence is a
287 base pair insertion/deletion polymorphism referred to as "ACE
I/D." In a normal diploid eukaryote, each gene has two loci, i.e.,
one gene copy at the same locus (position) on each of two matched
chromosomes. Different versions of a gene can occur at any locus,
and these versions are called alleles. Each allele may be the
wild-type (normal) allele or an allelic variant. Thus, two
different versions of a ACE gene will be present in any particular
subject's genome.
[0016] By "allelic variant" is meant a variation in a nucleotide
sequence, such as a single nucleotide polymorphism (SNP), a
multiple nucleotide polymorphism, or any other variant nucleic acid
sequence or structure (e.g., duplications, deletions, inversions,
insertions, translocations, etc.) in a gene that alters the
activity and/or expression of the gene, or correlates with the
occurrence of a disease or unhealthy state. Allelic variants can
over- or under-express the polypeptide encoded by the gene, and/or
express proteins with altered activities by virtue of having amino
acid sequences that vary from wildtype sequences. Allelic variants
need not occur in a coding sequence since variants at non-coding or
nonsense sequences also can correlate with the occurrence of a
disease or unhealthy state.
[0017] By "flanking primers" is meant one or more primers that
hybridize at either side of a nucleic acid sequence of interest,
but not within the sequence of interest itself. Such primers serve
as the starting points of nucleic acid replication by a DNA
polymerase, e.g., in an amplification reaction such as PCR.
[0018] In the present invention, a preferred allelic variant is a
polymorphism of the ACE gene, which is the presence or absence of
the 287 base pair nonsense DNA domain within Intron 16. Thus, a
"first ACE gene variant" may represent, e.g., the "I" variant
containing the 287 base pair nonsense DNA domain. Similarly, a
"second ACE gene variant" may represent the "D" variant that does
not contain the 287 base pair nonsense DNA domain. As discussed
above, the three potential ACE genotypes associated with this 287
base pair sequence are known to those of skill in the art as I/I
(for insertion/insertion; that is, each allele contains the
sequence); I/D (for insertion/deletion; that is, one allele
contains the insertion and one does not); and D/D (for
deletion/deletion; that is neither allele contains the insertion).
See, e.g., Winkelmann et al., "Pharmacogenomics and Complex
Cardiovascular Diseases--Clinical Studies in Candidate Genes," in
Pharmacogenomics, Licinio and Wong, eds pp. 254-61, Wiley-VCH,
2002.
[0019] The definitions above are not meant to exclude allelic
variants that have yet to be discovered and which correlate with a
disease or unhealthy state. The person of ordinary skill will
realize that other allelic variants are possible and may be used in
a similar manner.
[0020] Often, more than one allelic variant exists and persists in
a population of individuals. By "exist and persist" it is meant
that the frequency of incidence of a rare allele(s) is greater than
can be explained by recurrent mutation alone (i.e., typically
greater than 1%). However, the frequency of any variant allele may
vary over time due to such factors as genetic drift and the like.
When two or more different alleles of a gene are present in a
population, the gene or the protein it encodes is said to be
polymorphic. As used herein, a "polymorphism" refers to a specific
form of a gene or protein.
[0021] As used herein, the numeric order of a sequence is assigned
to the antisense strand of the sequence from 5' to 3' in increasing
number. A "forward primer" as used herein is a primer whose
sequence of nucleotides corresponds to a sequence this antisense
strand; a "reverse primer" is a primer whose sequence corresponds
to the complement of this strand (i.e., is of the same sense as the
sense strand of the gene). The skilled artisan will understand that
the designation of a primer as being "forward" or "reverse" is
arbitrary, but that a "forward" primer in a primer pair will
initiate DNA synthesis on one strand of the target DNA towards the
"reverse" primer, while a "reverse" primer will initiate DNA
synthesis on the complementary strand of the target DNA towards the
"forward" primer.
[0022] The "target sequence" is a site where the primer hybridizes
to the DNA and provides a site for DNA synthesis to begin by one or
more DNA polymerases.
[0023] By "DNA synthesis being initiated by the primers" is meant
that DNA synthesis begins at the site where a primer hybridizes
with a strand of DNA and provides a start point for a DNA
polymerase to begin DNA synthesis in an amplification reaction.
[0024] By "amplicon" is meant one or more copies of a nucleic acid
sequence that has been amplified by an amplification method such as
PCR.
[0025] The methods described herein are discussed in reference to
polymerase chain reaction ("PCR") amplification of genomic
sequences. The skilled artisan will understand, however, that
numerous methods are known in the art for amplification of nucleic
acids, and that these methods may be used either in place of, or
together with, the disclosed PCR steps. Nucleic acid amplification
methods, such as PCR, isothermal methods, rolling circle methods,
etc., are well known to the skilled artisan. See, e.g., Saiki,
"Amplification of Genomic DNA" in PCR Protocols, Innis et al.,
Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharam et
al., Nucleic Acids Res. 2001 Jun. 1; 29(11):E54-E54; Hafner et al.,
Biotechniques 2001 April; 30(4):852-6, 858, 860 passim; Zhong et
al., Biotechniques 2001 April; 30(4):852-6, 858, 860 passim.
[0026] The teen "biological sample" as used herein refers to a
sample obtained from a biological source, e.g., an organism, cell
culture, tissue sample, etc. A biological sample can, by way of
non-limiting example, consist of or comprise blood, sera, urine,
feces, epidermal sample, skin sample, cheek swab, sperm, amniotic
fluid, cultured cells, bone marrow sample and/or chorionic villi.
The DNA samples used in the methods described herein can be taken
from any source, but in preferred embodiments is from one of the
following sources: the endothelium of blood vessels, epithelial
cells, blood mononuclear cells, macrophages, and a biological
fluid.
[0027] The term "subject" as used herein refers to any eukaryotic
organism. Preferred subjects are fungi, plants, invertebrates,
insects, arachnids, fish, amphibians, reptiles, birds, marsupials
and mammals. A mammal can be a cat, dog, cow, pig, horse, ox,
elephant, or simian. Most preferred subjects are humans. A subject
can be a patient, which refers to a human presenting to a medical
provider for diagnosis or treatment of a disease. The term
"animals" includes prenatal forms of animals, such as fetuses.
[0028] As used herein, a "plurality of samples" refers to at least
two. Preferably, a plurality refers to a relatively large number of
samples. A plurality of samples is from about 5 to about 500
samples, preferably about 25 to about 200 samples, even more
preferably from about 50 to about 200 samples, and most preferably
from about 50 to about 100 samples. The term "about" in this
context refers to +/-10% of a given number. Samples that are
processed in a single batch run of the method of the invention are
usually prepared in plates having 24, 48, 96, 144, or 192 wells.
The term "samples" includes samples per se as well as controls,
standards, etc. that are included in a batch run.
[0029] Many embodiments of the methods are possible. In preferred
embodiments, the method includes the performance of a single
polymerase chain reaction amplification, the subject is a human,
and the sample is a human sample. In preferred embodiments, the
genotype is described as one of the following: insertion/insertion,
insertion/deletion, or deletion/deletion. In particularly preferred
embodiments, the genotype is determined by detecting the presence
of a polymorphism that resides on Intron 16 of chromosome 17q23,
and the polymorphism is the presence or absence of a 287 base pair
nonsense DNA domain.
[0030] In various preferred embodiments, the nucleic acid products
that are detected are a 157 base pair nucleic acid fragment and a
410 base pair nucleic acid fragment, indicating that the genotype
is I/I; a 123 base pair fragment, a 157 base pair nucleic acid
fragment, and a 410 base pair nucleic acid fragment, indicating
that the genotype is I/D; and a 123 base pair nucleic acid
fragment, indicating that the genotype is D/D. Thus, in the
preferred embodiments the genotype is determined by detecting the
presence or absence of these nucleic acid products. In other
embodiments where different primers are used, the nucleic acid
products that are detected are an approximately 157 base pair
nucleic acid fragment and an approximately 410 base pair nucleic
acid fragment, indicating that the genotype is I/I; an
approximately 123 base pair fragment, an approximately 157 base
pair nucleic acid fragment, and an approximately 410 base pair
nucleic acid fragment, indicating that the genotype is I/D; and an
approximately 123 base pair nucleic acid fragment, indicating that
the genotype is D/D. By "approximately" is meant plus or minus 10%.
In another embodiment the strands correspond to the stated lengths
plus or minus 20%.
[0031] In another aspect, the invention provides substantially
purified nucleic acids that may be used for amplifying DNA from the
ACE region of a DNA sample. The nucleic acids include one or more
of the following: a nucleic acid having the sequence 5'-CCA TCC TTT
CTC CCA TTT CTC T-3' (SEQ ID NO: 1); a nucleic acid having the
sequence 5'-GGA TGG TCT CGA TCT CCT GA-3' (SEQ ID NO: 2); and a
nucleic acid having the sequence 5'-CCT TAG CTC ACC TCT GCT TGT
AA-3' (SEQ ID NO: 3).
[0032] One or more nucleic acids of the set can be labeled with a
detectable "tag" or identifying reagent. In preferred embodiments,
one or more primers, most preferably the primer set forth in SEQ ID
NO: 3 is labeled with a detectable label, in preferred embodiments
at the 5' end with 6-FAM (fluorescein) or another fluorescent
reagent. Preferably, the present invention provides two, and most
preferably, each of the three nucleic acids in a single container
or environment, such as an aqueous solution.
[0033] Preferably, a nucleic acid for use as a primer in the
present invention binds in a complementary fashion to a portion of
a nucleic acid sequence that correlates with the ACE genotype
present in the sample, or that will be extended into such a
sequence by primer extension. Primers must be of a length
sufficient to provide specific binding to the target sequence of
interest. Such primers comprise an exact complement to the sequence
of interest for 15 to 75 nucleotides in length, preferably 17 to 50
nucleotides in length, and more preferably from 20 to 30
nucleotides in length.
[0034] As used herein, the term "purified" in reference to
oligonucleotides does not require absolute purity. Instead, it
represents an indication that the sequence is relatively more pure
than in the natural environment. Such oligonucleotides may be
obtained by a number of methods including, for example, laboratory
synthesis, restriction enzyme digestion or PCR. A "purified"
oligonucleotide is preferably at least 10% pure. A "substantially
purified" oligonucleotide is preferably at least 50% pure, more
preferably at least 75% pure, and most preferably at least 95%
pure.
[0035] In another aspect the present invention provides kits for
determining the genotype for angiotensin converting enzyme (ACE) in
a mammal. The kits include one or more substantially pure nucleic
acids of the invention, optionally include one or more solvents
and/or reagents useful in conducting assays to determine the
genotype for angiotensin converting enzyme (ACE), and optionally
include one or more containers for conducting assays and/or mixing
the reagents. Preferably, such kits contain primers in an amount
sufficient to perform an assay on at least one sample for
determining the ACE genotype in the sample, and more preferably in
an amount sufficient to perform an assay on a plurality of samples.
In preferred embodiments, the elements of the kit are contained in
an enclosure, such as a wrapping or box. In certain other
embodiments, the kits may also contain an instruction manual
providing instructions for use of the primers or other kit
materials in conducting the assays.
[0036] In various aspects of the present invention, the genotyping
methods described herein may be used to identify subjects at risk
for diseases such as essential hypertension, diabetic neuropathy,
renal disease, congestive cardiomyopathies including congestive
heart failure, and myocardial infarction, or at risk for adverse
outcomes from treatment, such as stent restenosis following
treatment for coronary artery disease, or poor (or heightened)
responsiveness to pharmaceutical intervention, such as treatment
with ACE inhibitors (such as benazepril, captopril, cilazapril,
enalapril, enalaprilat, fosinopril, lisinopril, moexipril,
perindopril, quinapril, ramipril, and trandolapril) or angiotensin
II type 1 receptor antagonists (such as irbesartan, losartan,
valsartan, telmisartan, camdesartam, and eprosartan). In these
aspects, the genotype of a subject is determined, and the
predisposition to disease or the risk of an adverse outcome
associated with that genotype is assigned to the subject.
[0037] In various preferred embodiments, the invention provides
methods of identifying a patient with a heightened risk of
suffering from a disease selected from the following: myocardial
infarction, ischemic and idiopathic dilated cardiomyopathy, sudden
death in hypertrophic cardiomyopathy, and restenosis after
percutaneous transluminal coronary angioplasty, based upon the
identified ACE genotype of the subject. By "heightened risk" is
meant that the patient's medical risk of suffering from a
particular disease increased relative to the general population. In
preferred embodiments, the heightened risk is at least one standard
deviation, more preferably two standard deviations, and most
preferably three standard deviations greater than that present in
the general population.
[0038] In various other preferred embodiments, the invention
provides a method for selecting a treatment regimen for a
particular subject, based upon the identified ACE genotype of the
subject. A "treatment regimen" is a course of treatment that may
include, but is not limited to, drug therapy, changes to lifestyle,
changes to diet, surgical intervention, installation of shunts,
.beta.-blockers (such as betaxolol and metipranolol), prostaglandin
analogs, osmotic diuretics, and combinations of these or other
treatments.
[0039] In yet other preferred embodiments, the invention provides a
method for selecting one or more subjects for inclusion in a
clinical trial, based upon the identified ACE genotype of the
subject(s). In these embodiments, subjects may be excluded or
included from the trial, according to their heightened risk of
suffering from a disease and/or their predicted responsiveness to a
particular treatment regimen.
[0040] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the invention, and from
the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0041] FIG. 1 provides a schematic representation of the genotyping
assays described herein, with reference to identification of a
genotype related to a deletion/insertion variant. The amplification
primers are shown as arrows, with the direction of the arrow
indicating the direction of primer extension.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention provides methods and compositions for
accurately determining the genotype of a gene of interest in a
single amplification reaction and with a minimum of required
reagents. As described in detail herein, preferably a primers pair
is employed that hybridize to nucleic acid sequences that flank a
variant-specific gene sequence. The variant-specific gene sequence
is preferably a 287 base pair insertion/deletion polymorphism in
the ACE gene that is a nonsense DNA domain within Intron 16 of the
ACE gene, the presence or absence of which correlates with the ACE
I/D genotype. In addition to the two flanking primers, a third
primer is also used. The third primer hybridizes within the
variant-specific sequence and forms a second pair of primers with
one of the flanking primers. The nucleic acid products of the
amplification are produced, detected, and correlated to a genotype.
FIG. 1 provides a schematic depiction of the amplification scheme
described above.
[0043] The human ACE intron 16 nucleic acid sequence (antisense)
for the I variant is as follows (SEQ ID NO: 4). An exemplary
forward primer sequence (SEQ ID NO: 1) is underlined italic, while
the complement to an exemplary reverse primer (SEQ ID NO: 3) is
underlined bold:
TABLE-US-00001 1 gtgagagctc atgtgcaggc tgagtgagag gcgagggctg
ggactggcat ggggcccggg 61 ggtgctgggt gagagcacag agttgggctc
ccctcgctct tggggtcagc gtgcccagga 121 aatgcccttt cttgttttcc
acgagggggg cttctctgcc cactgagagc cggcacctac 181 ttcataccat
gccccgatca gctgcccctc cctcagaacc gccctctgct taagggtgtc 241
cactctctcc tgtcctctct gcatgccgcc cctcagagca gcgggatctc aaagttatat
301 ttcatgggct tggactccaa atggggggaa ctcggggaca ctagctcccc
ccggcctcct 361 ttcgtgaccc tgcccttgac ttcctcacct tctctgtctt
tcctgagccc ctctcccagc 421 atgtgactga taaggaaatt gagtcacaca
gcccctgaaa gcgccagact agaacctgag 481 cctctgattc ctctcacttc
cctcccctac cctgccactt cctactggat agaagtagac 541 agctcttgac
tgtcctcttt tctccccact ggctggtcct tcttagcccc agcccgtttg 601
aaagagctca cccccgacac aaggacccgc acacagatac ctcccagctc cctctcaacc
661 caccctttcc agggttggag aacttgaggc ataaacattc ttccatgagg
aatctccacc 721 cagaaatggg tctttctggc ccccagccca gctcccacat
tagaacaatg acaaatagaa 781 ggggaaatgg aaaataaaca ggagaaacgg
ttttcccagg acagggtttg gcctacaagt 841 tgtggatgtg ggtacccatg
ccaagtgtga ggggaggctg gccgggtgtg gtggctcatg 901 ctctaatccc
agcactttgg gaggccaagg tgagtagatc acttgaggcc gggagtttga 961
gaccagcctg gccaacatgg tgaaacccca tctgtactaa aaatacaaaa gttagctggg
1021 cgtggtggta gatgcctgta gtcccagcta cttgggaggc tgaggcatga
gaatcgcttg 1081 agcccagcca gggcaataca gcaagacccc gtctctacaa
ataaaataca aaaaattagt 1141 tggatgtggt ggtgcatgcc tgtagtccta
gctgctaggg aggctgagat ggaaggattg 1201 cttgagcctg ggaggtcaag
gctgcagtga gccgagatgg cgccactgca ctccagcctg 1261 ggcaacagag
tgagaccctg tctcagaaag aaaaaaaaaa aaaaaggaga ggagagagac 1321
tcaagcacgc ccctcacagg actgctgagg ccctgcaggt gtctgcagca tgtgcccagg
1381 ccggggactc tgtaagccac tgctggagac cactcccatc ctttctccca
tttctctaga 1441 cctgctgcct atacagtcac tttttttttt tttttgagac
ggagtctcgc tctgtcgccc 1501 aggctggagt gcagtggcgg gatctcggct
cactgcaacg tccgcctccc gggttcacgc 1561 cattctcctg cctcagcctc
ccaagtagct gggaccacag cgcccgccac tacgcccggc 1621 taattttttg
tatttttagt agagacgggg tttcaccgtt ttagccggga tggtctcgat 1681
ctcctgacct cgtgatccgc ccgcctcggc ctcccaaagt gctgggatta caggcgtgat
1741 acagtcactt ttatgtggtt tcgccaattt tattccagct ctgaaattct
ctgagctccc 1801 cttacaagca gaggtgagct aagggctgga gctcaagcca
ttcaaccccc taccag
[0044] The "D" variant represents the loss of an alu-type sequence
from this intron in the region from nucleotide 1451 to nucleotide
1738 of the foregoing sequence. This region has the following
sequence (SEQ ID NO: 5). An exemplary forward primer (SEQ ID NO: 2)
for use in a primer pair with the exemplary reverse primer shown
above is underlined bold:
TABLE-US-00002 1 atacagtcac tttttttttt tttttgagac ggagtctcgc
tctgtcgccc aggctggagt 61 gcagtggcgg gatctcggct cactgcaacg
tccgcctccc gggttcacgc cattctcctg 121 cctcagcctc ccaagtagct
gggaccacag cgcccgccac tacgcccggc taattttttg 181 tatttttagt
agagacgggg tttcaccgtt ttagccggga tggtctcgat ctcctgacct 241
cgtgatccgc ccgcctcggc ctcccaaagt gctgggatta caggcgtg
[0045] Amplification Reaction
[0046] The methods described herein are discussed in reference to
polymerase chain reaction ("PCR") amplification of genomic
sequences. As noted above, the skilled artisan will understand that
numerous methods are known in the art for amplification of nucleic
acids, and that these methods may be used either in place of, or
together with, the disclosed PCR steps.
[0047] The objective of PCR is to amplify a specific DNA fragment,
referred to as the "target sequence." Primers function in pairs, a
so-called forward primer and a so-called reverse primer, with this
distinction being arbitrary from the computational viewpoint. The
primer pairs are chosen such that primer extension occurs towards
one another to cover a given target region. PCR begins with a high
temperature (95.degree. C.) denaturation step converting the
double-stranded DNA into single-stranded DNA, followed by a low
temperature step (45-65.degree. C.) during which the primers
hybridize and finally an intermediate temperature step (72.degree.
C.) for the primer extension. Typically 25-45 of these cycles are
performed.
[0048] Formally, primers are considered as strings over the
alphabet .SIGMA.{A, C, G, T} with the set of all these strings
being .SIGMA.*. As described herein, the first position of a primer
indicates the 5' end while the terminating position indicates the
3' end. Each primer is chosen within a window whose length and
location is subject to the discretion of the skilled artisan.
[0049] Primer assessment extends beyond string matching and
involves criteria including the proximity between primer melting
temperatures, minimization of hybridization effects between forward
and reverse primers, and avoidance of hybridization of primers with
themselves. The latter two criteria are dealt with by annealing
values. The design complexity increases in so-called multiplex PCR.
This involves performing multiple PCR reactions simultaneously in a
single tube. Consequently, this requires that physical parameters
such as cycle number, cycle duration and annealing temperature are
identical for all of the PCR reactions. Additional information
regarding the design of other primers is found at "Efficient primer
design algorithms," Bioinformatics 17: 214-225 (2001).
[0050] For each reaction mixture, the amount of the nucleic acid
sufficient for primer extension can be determined by obtaining a
sample comprising nucleic acid and determining the concentration of
nucleic acid therein. One skilled in the art will be able to
prepare such samples to a concentration and purity necessary to
practice the invention, and to estimate the amount of a specific
sample that should be added to a particular reaction mixture. A
failure to detect a signal in the method of the invention may
signify that, among other things, an inadequate amount of nucleic
acid has been added to a reaction mixture. Those skilled in the art
will be able to trouble-shoot failed batch runs and adjust the
contents of the reaction mixtures and/or conditions of the run
accordingly. Control samples, both positive and negative, can be
included in the batch runs to confirm that appropriate amounts of
nucleic acid are present.
[0051] In the exemplary embodiments of the invention described
below, three primers are used--two forward primers and one reverse
primer. Forward primer 1 (SEQ ID NO: 1), which flanks the region of
interest; forward primer 2 (SEQ ID NO: 2), which is specific for
the insertion; and a reverse primer (SEQ ID NO: 3), which flanks
the region of interest. Thus, the two forward primers share one
reverse primer. In alternative embodiments, two reverse primers can
be used with one forward primer without changing the principles of
the invention described herein.
[0052] Applicants have determined that the "three primer, two
primer pair" methods described herein can provide sensitive and
specific detection of the ACE genotype present in a sample without
the generation of spurious PCR products that increase the
background signal obtained. It has thus been discovered
unexpectedly by the inventors that utilizing a single PCR reaction
with the elimination of one reverse primer results in clearer, more
easily interpretable results, and the elimination of a second PCR
reaction. The invention advantageously provides detection of a
polymorphism in the ACE gene based on the presence or absence of a
287 base pair nonsense DNA domain within Intron 16.
[0053] The genotypes relating to the invention can be described as
I/I, I/D, and D/D. The I/I (insertion/insertion) genotype indicates
a subject having two alleles containing the 287 base pair insertion
within Intron 16; the I/D (insertion/deletion) genotype indicates a
subject having one allele containing the insertion and the other
allele lacking the insertion; and the D/D genotype indicates a
subject having no alleles containing the insertion.
[0054] Detection of Amplification Products
[0055] Following amplification, the resulting nucleic acid products
can then be denatured (e.g., in formamide) and fractionated for
detection of the products generated, e.g., by capillary
electrophoresis ("CE"). In the exemplary methods described
hereinafter, an ABI PRISM.RTM. 310 Genetic Analyzer (Applied
Biosystems) is used with ABI PRISM.RTM. 310 Genetic Analyzer
capillaries (47 cm.times.50 um capillaries). For reviews of the use
of CE in DNA sequencing and polymorphism analysis, see Heller,
Electrophoresis 22:629-43, 2001; Dovichi et al., Meth. Mol. Biol.
167:225-39, 2001; Mitchelson, Methods Mol. Biol. 162:3-26, 2001;
and Dolnik, J. Biochem. Biophys. Meth. 41:103-19, 1999.
[0056] Examples of other apparatuses that may be useful for
electrophoresis and visualization are an agarose gel
electrophoresis apparatus, such as CBS Scientific horizontal
mini-gel; a power supply having a constant voltage of 100 to 200V
or better variable power supply for electrophoresis, such as the
BioRad Model 200; photodocumentation apparatus, such as the Alpha
Innotech AlphaImager or Polaroid DS34 t; and a transilluminator,
e.g., a VWR Model LM-20E or equivalent. Other methods of
fractionating the amplification products can also be utilized, such
as standard or HPLC chromatography methods, or nucleic acid
microarray hybridization.
[0057] The different genotype alleles are then detected based on
the nucleic acid fragment size. Thus, the following describes the
fragment sizes obtained using the exemplary primers descried
herein, and shows the corresponding genotype indicated:
TABLE-US-00003 Genotype PCR fragment (bp) I/I 157, 410 I/D 123,
157, 410 D/D 123
[0058] In preferred embodiments, the primer nucleic acid that is
common to both primer pairs in the "three primer, two primer pair"
methods is labeled with a detectable label in order to provide
labeled amplification products. In the alternative or together with
labeling the common primer, one or both of the non-common primer
nucleic acids may also be labeled, or one or more labeled
deoxynucleotide triphosphates may be incorporated into the
amplification reaction. Numerous detectable labels for
incorporation into nucleic acids are known to those of skill in the
art. See, e.g., Handbook of Fluorescent Probes and Research
Products, 9.sup.th ed., Molecular Probes, Inc., 2002, Chapter 8
("Nucleic Acid Detection and Genomics Technology"). Illustrative
fluorescent labels include xanthene dyes, naphthylamine dyes,
coumarins, cyanine dyes and metal chelate dyes, such as
fluorescein, rhodamine, rosamine, the BODIPY dyes (FL, TMR, and
TR), dansyl, lanthanide cryptates, erbium. terbium and ruthenium
chelates, e.g. squarates, and the like. Additionally, in certain
embodiments, one or more fluorescent moieties can be energy
transfer dyes such as those described in Waggoner et al., U.S. Pat.
No. 6,008,373. In the exemplary embodiments described hereinafter,
the reverse primer is labeled at the 5' end with 6-FAM (blue)
fluorescent dye.
[0059] In the exemplary embodiments described herein, the ACE gene
region contains a 287 by insertion/deletion polymorphism, which is
amplified by using a first forward primer (e.g., SEQ ID NO: 1) and
a reverse primer (e.g., SEQ ID NO: 3), each of which flanks the 287
by region. A fragment is also amplified from I/I and genotypes with
an insertion-specific second forward primer (e.g., SEQ ID NO: 2),
which forms a second primer pair with the flanking reverse primer.
The primers of the present invention can be manufactured using
common methods of nucleic acid synthesis. For example, an automated
nucleic acid synthesizer is preferred. In the most preferred
embodiment, the first forward primer has the sequence 5'-CCA TCC
TTT CTC CCA TTT CTC T-3' (SEQ ID NO: 1). The second forward primer
has the sequence 5'-GGA TGG TCT CGA TCT CCT GA-3' (SEQ ID NO: 2).
And the reverse primer is preferably labeled at the 5' end with a
detectable label (e.g., 6-FAM (fluorescein)) and has the sequence
5'-(6-FAM)-CCT TAG CTC ACC TCT GCT TGT AA-3' (SEQ ID NO: 3).
[0060] Identification of at-Risk Individuals
[0061] The ACE genotype, and particularly the genotype regarding
the I/D polymorphism, of an individual has been associated with
various diseases, including myocardial infarction, coronary artery
disease, ischemic and idiopathic dilated cardiomyopathy, sudden
death in hypertrophic cardiomyopathy, and restenosis after
percutaneous transluminal coronary angioplasty. Thus, the methods
and compositions of the present invention can be used to identify
such individuals in a clinical setting. In addition, identification
of such individuals can also be used for ruling in or out certain
individuals in various situations such as clinical trials and
prospective or retrospective clinical studies, where the
predisposition to a particular disease may be required for
inclusion, or indicative of exclusion, from a selected group of
individuals. The following is a list of exemplary diseases
associated with the ACE I/D polymorphism:
TABLE-US-00004 Genotype Disease of Risk Citation Cardiovascular
disease D/D Circulation 1998, 97: 1780-1783 Myocardial Ischemia and
Myocardial D/D J Am Coll Cardiol 1997, 29: 1468-73 infarction
Aoronary Artery Disease D/D J Investing Med 1995, 43: 275-280
Restenosis D/D Circulation 1997, 96: 56-60 Coronary Artery Disease
D/D Atherosclerosis, 1998, 139: 153-159 Myocardial Infarction D/D
Nature 1992, 359: 641-644 Coronary Atherosclerosis D/D Br Heart J
1995; 584-591 Stent Restenosis D/D Circulation 1998; 147-154
Coronary Heart Disease D/D Proc Natl Acad Sci USA 1994; 3662-3665
Essential Hypertension D/D Biochem Biophys Res Comm 1992; 9-15
Myocardial Infarction D/D Clin Genetic 1992; 46: 94-101
Atherosclerotic Plaque Calcification D/D JACC 1998; 31: 987-991
Essential Hypertension D/D Circulation 2000; 101: 2060-2065
Diabetic Nephropathy D/D J Diabetes Its Compl 2002; 16: 255-262
[0062] The ACE genotype of an individual has also been related to
response to various clinical treatment regimens. Thus, the methods
and compositions of the present invention can be used to identify
an appropriate treatment regimen for an individual. In addition,
identification of such individuals can also be used for ruling in
or out certain individuals in various situations such as clinical
trials and prospective or retrospective clinical studies, where
response to a particular treatment regimen is required for
inclusion, or indicative of exclusion, from a selected group of
individuals. The following is a list of exemplary treatments
associated with the ACE I/D polymorphism:
TABLE-US-00005 ACE Disease Drug Impact Genotype Citation Coronary
atherosclerosis Fluvastatin + D/D J Am Coll Cardiol 2000, 35: 89-95
Restenosis ACE Inhibitor + I/I Angiology 1999, 50: 811-822
(Imidapril) Congestive Heart Failure Beta-Blocker 0 D/D Circulation
2001; 103: 1644 Chronic Cardiac Failure ACE Inhibitor + D/D Ter
Arkh 2002; 74: 54-58 (Perindopril) Restenosis ACE Inhibitor + D/D
Circ J 2002; 66: 311-316 (Quinapril)
Example 1
[0063] This example describes one embodiment of reagents for
performing the present methods. The person of ordinary skill in the
art will realize that many variations of these reagents are
possible without departing from the invention, and those variations
are contemplated within the present invention.
[0064] PCR primers can be made according to standard and well-known
techniques, such as by using an automated nucleic acid synthesizer.
Primers can also be purchased commercially from various suppliers
(e.g., Operon Technologies, Inc., Alameda, Calif.). Amplification
primers for a particular gene are designed as follows. A first
primer pair, comprising a first and second primer, is designed to
flank a gene sequence, the presence of which indicates the presence
of a gene variant, and the absence of which indicates the presence
of a different gene variant. Each member of the first primer pair
is also designed to initiate transcription towards the other member
of the first primer pair. In this way, amplification will generate
an amplicon having one length when the gene sequence is present,
and a different length when the gene sequence is absent.
[0065] A third primer is designed to specifically bind only when
the variant-specific gene sequence is present, and to function as a
member of a second primer pair with the first or second primer.
Each member of the second primer pair is also designed to initiate
transcription towards the other member of the second primer pair.
In this way, amplification will generate an amplicon only when the
gene sequence is present.
[0066] The skilled artisan will acknowledge that such methods are
generally applicable to any gene present as a plurality of alleles
in a genome, in which the presence or absence of a specific
sequence within the gene is to be distinguished. In an exemplary
embodiment for identification of the ACE I/D polymorphism genotype,
three primers (SEQ ID NOS: 1-3) were used. The reverse primer (SEQ
ID NO: 3) was 5' labeled with 6-FAM (fluorescein).
[0067] An embodiment of a PCR "master mix" buffer was made
according to the following formula: 50 units/ml of Taq polymerase,
400 .mu.M each of dATP, dGTP, dCTP, and dTTP; and 3 mM
MgCl.sub.2.
[0068] An embodiment of loading mix was made by combining 23.9
.mu.L of deionized formamide with 0.1 .mu.L of Rox-500 for a total
of 24 ul per sample to be tested. GeneScan ROX-500HD Size Standard
can be obtained from commercial suppliers (Applied Biosystems).
Other appropriate size standards (e.g., TAMRA-labeled standards)
are also commercially available and known to those of ordinary
skill in the art.
[0069] In a preferred embodiment the samples were prepared for the
PCR reaction by combining 12.5 .mu.L of PCR master mix, 0.25 .mu.L
of forward primer 1 (10 .mu.M), 0.3 .mu.L, reverse primer (10
.mu.M), 0.125 .mu.L forward primer 2 (10 .mu.M), 1.25 .mu.L DMSO,
and 9.575 .mu.L of nuclease-free water. These amounts were per
sample to be tested, for a total of 24 .mu.L sample volume.
Example 2
[0070] This example describes determination of a genotype from a
DNA sample obtained from whole blood. Genomic DNA was extracted
from whole blood by standard methods. 24 .mu.L of PCR master mix
was pipetted into a 0.2 ml PCR tubes and 1 .mu.L of control samples
(3 positive and 2 negative) and patient DNA was added to their
respective tubes. The tubes were vortexed for about 5 seconds.
[0071] The samples were placed into a thermal cycler for PCR
amplification when the cycler temperature reached 85.degree. C. The
PCR cycles were performed as follows:
TABLE-US-00006 1 94.degree. C. 2 min 2 94.degree. C. 60 sec 3
58.degree. C. 60 sec 4 72.degree. C. 90 sec 5 go to step 2 29
cycles* 6 72.degree. C. 15 min 7 4.degree. C. hold *Typically 30
cycles is optimal, but 29-31 cycles may be used if amplified
products from 30 cycles are less than optimal.
[0072] After the PCR was complete, the samples were prepared for
size fractionation by capillary electrophoresis on a commercially
available Genetic Analyzer (ABI 310 Genetic Analyzer, Applied
Biosystems, Inc., Foster City, Calif.). A 96 well plate tray with
tubes was prepared. 24 .mu.L of loading mix was placed into each
sample tube with 1 .mu.L of PCR products. The samples were vortexed
briefly and placed in a 95.degree. C. heat block for 3 minutes, and
then immediately placed into a refrigerator for at least 3 minutes
or until use. The samples were size fractionated according to
manufacturer's instructions.
[0073] The person of ordinary skill in the art will realize that
many methods of size fractionation are available (e.g., HPLC,
manual gel electrophoresis, etc.) and that the samples can be size
fractionated according to any of these methods without departing
from the invention. Similarly, other methods besides size
fractionation can be designed to determine the presence or absence
of the indicating sequences and therefore yield the information
provided by the methods, and those methods are also contemplated as
being within the invention.
[0074] The various samples were then analyzed to correlate the PCR
products with an ACE genotype. In those samples where a 157 by
fragment and a 410 by fragment was present, and no 123 by fragment
was present, the genotype was assigned as insertion/insertion
(I/I); in those samples where a 123 by fragment, a 157 by fragment,
and a 410 by fragment were present, the genotype was assigned as
insertion/deletion (I/D); in those samples where only a 123 by
fragment is present, the genotype was assigned as deletion/deletion
(D/D):
TABLE-US-00007 TABLE 1 Potential PCR Results: Genotype PCR
Fragments Generated (base pairs) I/I 157, 410 I/D 123, 157, 410 D/D
123
[0075] The person of ordinary skill in the art will realize that
primers with other sequences can be designed and used according to
the invention and without departing from the invention. Thus, while
the terms "123 by fragment," "157 by fragment," and "410 by
fragment" are used in this application, the person of ordinary
skill in the art will realize that the number of base pairs in the
fragment is not exact and can vary depending on the exact primer
sequence used. Thus, for example, the "410 by fragment" may contain
a lesser or greater number of base pairs if amplified using a
different primer. The other fragments may also vary in their size
for the same reason. But what is important is that the fragments
can be identified and correlated to one of the genotypes described
above.
[0076] One or more of steps of the assays described herein, in any
combination, are preferably performed in an automated fashion,
typically using robotics, in order to provide for the processing of
a large number of samples in a single batch run. Preferred forms of
automation will provide for the preparation and separation of a
plurality of labeled nucleic acids in small volumes. The term
"small volumes" refers to volumes of liquids less than 2 ml, e.g.,
any volume from about 0.001 picoliters or about 0.001 .mu.l, to any
volume about 2 ml, 500 .mu.l, 200 .mu.l, 100 .mu.l, 10 .mu.l, 1
.mu.l, 0.1 .mu.l, 0.01 .mu.l, or 0.001 .mu.l. Additionally,
capillary electrophoresis of the resulting amplification products
is preferred over agarose gel electrophoresis for purposes of
automated and/or high-throughput applications.
[0077] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[0078] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[0079] In addition, where features or aspects of the invention are
described in teens of Markush groups, those skilled in the art will
recognize that the invention is also thereby described in terms of
any individual member or subgroup of members of the Markush
group.
[0080] Other embodiments are set forth within the following claims.
Sequence CWU 1
1
5122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ccatcctttc tcccatttct ct 22220DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
2ggatggtctc gatctcctga 20323DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 3ccttagctca cctctgcttg taa
2341856DNAHomo sapiens 4gtgagagctc atgtgcaggc tgagtgagag gcgagggctg
ggactggcat ggggcccggg 60ggtgctgggt gagagcacag agttgggctc ccctcgctct
tggggtcagc gtgcccagga 120aatgcccttt cttgttttcc acgagggggg
cttctctgcc cactgagagc cggcacctac 180ttcataccat gccccgatca
gctgcccctc cctcagaacc gccctctgct taagggtgtc 240cactctctcc
tgtcctctct gcatgccgcc cctcagagca gcgggatctc aaagttatat
300ttcatgggct tggactccaa atggggggaa ctcggggaca ctagctcccc
ccggcctcct 360ttcgtgaccc tgcccttgac ttcctcacct tctctgtctt
tcctgagccc ctctcccagc 420atgtgactga taaggaaatt gagtcacaca
gcccctgaaa gcgccagact agaacctgag 480cctctgattc ctctcacttc
cctcccctac cctgccactt cctactggat agaagtagac 540agctcttgac
tgtcctcttt tctccccact ggctggtcct tcttagcccc agcccgtttg
600aaagagctca cccccgacac aaggacccgc acacagatac ctcccagctc
cctctcaacc 660caccctttcc agggttggag aacttgaggc ataaacattc
ttccatgagg aatctccacc 720cagaaatggg tctttctggc ccccagccca
gctcccacat tagaacaatg acaaatagaa 780ggggaaatgg aaaataaaca
ggagaaacgg ttttcccagg acagggtttg gcctacaagt 840tgtggatgtg
ggtacccatg ccaagtgtga ggggaggctg gccgggtgtg gtggctcatg
900ctctaatccc agcactttgg gaggccaagg tgagtagatc acttgaggcc
gggagtttga 960gaccagcctg gccaacatgg tgaaacccca tctgtactaa
aaatacaaaa gttagctggg 1020cgtggtggta gatgcctgta gtcccagcta
cttgggaggc tgaggcatga gaatcgcttg 1080agcccagcca gggcaataca
gcaagacccc gtctctacaa ataaaataca aaaaattagt 1140tggatgtggt
ggtgcatgcc tgtagtccta gctgctaggg aggctgagat ggaaggattg
1200cttgagcctg ggaggtcaag gctgcagtga gccgagatgg cgccactgca
ctccagcctg 1260ggcaacagag tgagaccctg tctcagaaag aaaaaaaaaa
aaaaaggaga ggagagagac 1320tcaagcacgc ccctcacagg actgctgagg
ccctgcaggt gtctgcagca tgtgcccagg 1380ccggggactc tgtaagccac
tgctggagac cactcccatc ctttctccca tttctctaga 1440cctgctgcct
atacagtcac tttttttttt tttttgagac ggagtctcgc tctgtcgccc
1500aggctggagt gcagtggcgg gatctcggct cactgcaacg tccgcctccc
gggttcacgc 1560cattctcctg cctcagcctc ccaagtagct gggaccacag
cgcccgccac tacgcccggc 1620taattttttg tatttttagt agagacgggg
tttcaccgtt ttagccggga tggtctcgat 1680ctcctgacct cgtgatccgc
ccgcctcggc ctcccaaagt gctgggatta caggcgtgat 1740acagtcactt
ttatgtggtt tcgccaattt tattccagct ctgaaattct ctgagctccc
1800cttacaagca gaggtgagct aagggctgga gctcaagcca ttcaaccccc taccag
18565278DNAHomo sapiens 5atacagtcac tttttttttt tttttgagac
ggagtctcgc tctgtcgccc aggctggagt 60gatctcggct cactgcaacg tccgcctccc
gggttcacgc cattctcctg cctcagcctc 120ccaagtagct gggaccacag
cgcccgccac tacgcccggc taattttttg tatttttagt 180agagacgggg
tttcaccgtt ttagccggga tggtctcgat ctcctgacct cgtgatccgc
240ccgcctcggc ctcccaaagt gctgggatta caggcgtg 278
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