U.S. patent application number 10/773440 was filed with the patent office on 2005-12-22 for method to detect prostate cancer in a sample.
This patent application is currently assigned to Diagnocure Inc.. Invention is credited to Chypre, Camille, Fradet, Yves, Garon, Genevieve, Piche, Lyson.
Application Number | 20050282170 10/773440 |
Document ID | / |
Family ID | 32850986 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050282170 |
Kind Code |
A1 |
Fradet, Yves ; et
al. |
December 22, 2005 |
Method to detect prostate cancer in a sample
Abstract
The present invention relates to prostate cancer. More
specifically, the present invention relates to a method to detect
prostate cancer in a patient sample by detecting the RNA encoded by
the gene PCA3. More particularly the present invention relates to a
method for determining a predisposition, or presence of prostate
cancer in a patient comprising: (a) contacting a biological sample
of a patient with at least one oligonucleotide that hybridizes to a
PCA3 polynucleotide; (b) detecting in the biological sample an
amount of PCA3 and second prostate specific polynucleotides; and
(c) comparing the amount of PCA3 polynucleotide that hybridizes to
the oligonucleotide to a predetermined cut off value, and therefrom
determining the presence or absence of prostate cancer in the
biological sample. The present invention further relates to
diagnostic kits for the detection of prostate cancer or the risk of
developing same in a patient comprising: (a) at least one container
means having disposed therein at least one oligonucleotide probe or
primer that hybridizes to one a PCA3 nucleic acid or complement
thereof; (b) at least one oligonucleotide probe or primer that
hybridizes with a second prostate specific nucleic acid or
complement thereof; and (c) reagents enabling a detection of PCA3
and of the second prostate specific nucleic acid when PCA3 or
second prostate-specific nucleic acid sequence is present.
Inventors: |
Fradet, Yves; (Sillery,
CA) ; Chypre, Camille; (Sillery, CA) ; Piche,
Lyson; (Cap-Sante, CA) ; Garon, Genevieve;
(Saint-Augustin-de-Desmaures, CA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Diagnocure Inc.
|
Family ID: |
32850986 |
Appl. No.: |
10/773440 |
Filed: |
February 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60445436 |
Feb 7, 2003 |
|
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|
Current U.S.
Class: |
435/6.14 |
Current CPC
Class: |
C12Q 2600/16 20130101;
C12Q 1/6886 20130101; C12Q 2600/158 20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for determining a predisposition, or presence of
prostate cancer in a patient comprising: a) contacting a biological
sample of said patient with at least one oligonucleotide that
hybridizes to a PCA3 polynucleotide selected from the group
consisting of: i) a polynucleotides according to SEQ ID NOs 9, 10
and 13; ii) a polynucleotide sequence that hybridizes under high
stringency conditions to the polynucleotide sequence in i); and
iii) a polynucleotide sequence fully complementary to i) or ii);
and contacting said biological sample with at least one
oligonucleotide that hybridizes with a second prostate specific
polynucleotide, b) detecting in said biological sample an amount of
PCA3 and second prostate specific polynucleotides; and comparing
the amount of PCA3 polynucleotide that hybridizes to the
oligonucleotide to a predetermined cut off value, and therefrom
determining the presence or absence of prostate cancer in the
biological sample.
2. The method of claim 1, wherein said second specific prostate
specific nucleic acid is selected from the group consisting of:
PSA, human kallikrein 2, PSMA, transglutaminase 4, acid phosphatase
and PCGEM1 nucleic acid.
3. The method of claim 2, wherein said prostate specific nucleic
acid is PSA.
4. The method of claim 3, wherein said PSA sequence hybridizes to
human kallikrein 2.
5. The method of claim 1, wherein the amount of PCA3 polynucleotide
and of the second specific prostate cancer polynucleotide is
determined using an assay selected from the group consisting of: a)
an amplification assay; and b) a hybridization assay.
6. The method of claim 5, wherein said amplification assay is an in
vitro RNA amplification method.
7. The method of claim 6, wherein said RNA amplification method is
selected from the group consisting of: a) nucleic acid
sequence-based amplification (NASBA); b) polymerase chain reaction
(PCR); c) transcription mediated amplification assay (TMA); and d)
ligase chain reaction.
8. The method of claim 6, wherein said amplification of PCA3 and
said second prostate specific nucleic acid is performed
simultaneously.
9. The method of claim 6, wherein said amplification of PCA3 is
carried out using a primer pair composed of SEQ ID NOs: 3 and
4.
10. The method of claim 6, wherein said detection is performed by
fluorescence, chimiluminescence or colorimetry detection.
11. The method of claim 10, wherein said detection of PCA3 is
carried out using acridinium ester compounds.
12. The method of claim 6, wherein said detection of PCA3 is
carried out using a molecular beacon.
13. The method of claim 12, wherein said beacon has the sequence
set forth in SEQ ID NO: 6.
14. The method of one of claims 6, wherein said second prostate
specific nucleic acid is PSA and said amplification thereof is
carried out using a primer pair composed of SEQ ID NOs: 1 and
2.
15. The method of claim 14, wherein said detection of PSA is
carried out using acridinium ester compounds.
16. The method of claim 14, wherein said detection of PSA is
carried out using a PSA molecular beacon.
17. The method of claim 16, wherein said PSA beacon has the
sequence set forth in SEQ ID NO: 5.
18. The method of claim 1, wherein said sample contains at least
one prostate cell and said at least one cell is collected from said
sample prior to step a).
19. The method of claim 18, wherein said nucleic acid is extracted
from said at least one prostate cell.
20. The method of claim 19, wherein said nucleic acid is RNA.
21. The method of claim 20, wherein said RNA is extracted using a
silica-based method.
22. The method of claims 1, wherein said sample is selected from
the group consisting of: a) urine; b) blood or fraction thereof;
and c) prostate biopsy.
23. The method of claim 22 wherein said sample is urine.
24. The method of claim 23, wherein said urine is collected
following a digital rectal examination, thereby increasing the
number of prostate cells in said sample.
25. The method of claim 1, further comprising: c) repeating steps
(a) and (b) using a biological sample from the patient at a
subsequent point in time; and d) comparing the relative amount of
said PCA3 polynucleotide detected in step (c) to the relative
amount of PCA3 polynucleotide detected in step (b) and therefrom
monitoring the progression of the prostate cancer in the
patient.
26. The method of claim 1, wherein the detection of the second
prostate specific polynucleotide validates a negative result for
PCA3 detection.
27. The methods of claim 1, wherein the biological sample is spiked
with an internal control IC selected from the group consisting of:
a) purified nucleic acid; b) cells; c) viral particules containg
target nucleic acids; and d) organelles.
28. The method of claim 6, wherein RNA is extracted using a target
capture method.
29. The method of claim 1, wherein said detection of PCA3 is
carried out using chemiluminescent labels in a homogenous detection
method.
30. A diagnostic kit for the detection of prostate cancer or the
risk of developing same in a patient comprising: a) at least one
container having disposed therein at least one oligonucleotide
probe or primer that hybridizes to one of: i) a PCA3 nucleic acid
sequence according to SEQ ID NO: 9, 10 and 13; ii) a sequence which
is fully complementary to i); and iii) a sequence which hybridizes
under high stringency conditions to i) or ii); b) at least one
oligonucleotide probe or primer that hybridizes with a second
prostate specific nucleic acid or complement thereof; and c)
reagents enabling a detection of PCA3 and of said second prostate
specific nucleic acid when said PCA3 or second prostate-specific
nucleic acid sequence is present.
31. The diagnostic kit according to claim 30, wherein the detection
reagent comprises a reporter group or label selected from the group
consisting of: a) radioisotopes; b) enzymes; c) fluorescent groups;
d) biotin; e) chemiluminescent groups; and f) dye particles.
32. The kit of claim 30, wherein said PCA3 nucleic acid and said
second prostate specific nucleic acid are amplified simultaneously
in the same container.
33. The kit of claim 30, wherein the detection of said PCA3 nucleic
acid and said second prostate specific nucleic acid is performed in
the same container.
34. The kit of claim 30, further comprising an internal control
(IC) as well as a primer, and/or probe, and/or reagent for the
amplification, and/or hybridization, and/or detection of said
internal control.
35. The kit of claim 34, wherein said IC is selected from the group
consisting of: a) purified nucleic acid; b) cells; c) viral
particules containg target nucleic acids; and d) organelles.
36. A kit for assessing the presence of prostate cancer or the risk
of developing same in a patient comprising: a) a first primer pair
specific for amplifying a PCA3 nucleic acid associated with
prostate cancer present in a patient sample; b) a second primer
pair specific for amplifying a second prostate-specific nucleic
acid; and c) reagents enabling a detection of PCA3 and of said
second prostate specific nucleic acid amplification products when
said PCA3 or second prostate-specific nucleic acid sequence is
present.
37. A method for detecting prostate cancer in a human patient,
comprising: a) performing an in vitro nucleic acid amplification
assay on a biological sample of said patient or extract thereof
using a first primer pair which is specific to a prostate cancer
specific PCA3 sequence and a second primer pair which is specific
to a prostate specific nucleic acid sequence; and b) detecting said
PCA3 sequence and said prostate specific nucleic acid sequence,
wherein, a detection of said PCA3 nucleic acid sequence or a level
thereof correlates with a risk of developing prostate cancer or to
a presence of prostate cancer in said patient, and wherein an
absence of detection of said PCA3 nucleic acid sequence or lower
level thereof in said sample validates an absence of prostate
cancer or a lower risk of developing same, when said second
prostate specific nucleic acid is detected.
38. The method of one of claim 1, wherein said nucleic acid
amplification is carried-out in real time.
39. The method of claim 37, wherein said detection is performed by
fluorescence, chimiluminescence or colorimetry detection.
40. The method of claim 8, wherein said amplification of PCA3 and
said second prostate specific nucleic acid is performed
simultaneously in one container.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to prostate cancer. More
specifically, the present invention relates to a method to detect
prostate cancer in a patient sample by detecting an RNA encoded by
the prostate cancer antigen PCA3 gene.
BACKGROUND OF THE INVENTION
[0002] Over the last decade, cancer of the prostate has become the
most commonly diagnosed malignancy among men and the second leading
cause of male cancer deaths in the western population, following
lung cancer.
[0003] Early detection and treatment of prostate cancer before it
has spread from the prostate gland, reduces the mortality of the
disease. This is particularly true for younger men who are at
greater risk of dying from this pernicious but slowly growing
malignancy. This realization has prompted increasing efforts for
early diagnosis and treatment. Indeed, the American Cancer Society
and the American Urological Association recommend that male
population at large undergo annual screening for prostate cancer
beginning at age 50. The recommended age for screening is lowered
to 40 for men giving a family history of prostate cancer or other
risk factors.
[0004] With this increasing focus on prostate cancer screening,
more men than ever before are being routinely tested for prostate
cancer. Not surprisingly, this practice has increased early
detection of onset of the disease, as reflected by an apparent
increase in the incidence of prostate cancer and decrease in the
apparent average age of diagnosis. The clinical hope is that
earlier detection of prostate cancer before it metastasizes will
reduce the overall mortality rate. Healthcare payers look for early
screening and detection to translate into a reduction in the
healthcare burden, as early treatment can be less radical, more
successful and therefore provided at a lower cost per treated
patient. The key to accomplishing this goal remains providing
better differential diagnostic tools.
[0005] Screening for prostate cancer now involves both palpation of
the prostate by digital rectal examination (DRE) and assay of
plasma levels of prostate specific antigen (PSA/hK3/hKLK3). PSA is
a serine protease produced by the prostatic epithelium that is
normally secreted in the seminal fluid to liquefy it. Disruption of
the anatomic integrity of the prostate gland can compromise the
cellular barriers that normally restrict PSA to within the duct
system of the prostate, allowing it to disperse into blood or
urine. A number of conditions can result in leakage of PSA into the
blood. They include inflammation of the prostate, urinary
retention, prostatic infection, benign prostatic hyperplasia (BPH),
and prostate cancer. Physical manipulation of the prostate can also
increase serum PSA levels, but a mild stimulus, such as digital
rectal examination (DRE), does not normally increase serum PSA. It
is therefore not surprising that screening of serum PSA as an
indicator of prostate cancer is not absolutely predictive.
[0006] Despite the fact that measure of blood PSA levels can be the
result from a variety of different causes, it is nonetheless the
basis for primary screening for prostate cancer. Measurement of
total PSA (tPSA) as a diagnostic assay to predict prostate cancer
has been in use since 1991. Levels of 4 ng/ml or greater in blood
serum are considered abnormal and predictive of prostate cancer.
However, the sensitivity of such elevated tPSA levels is only 79%;
thus leaving 21% of patients with prostate cancer undetected. The
specificity for all tPSA values of 4 ng/ml or greater is very poor.
In addition, estimates of specificity for tPSA levels >4.0 ng/ml
are reported to be in the range of 20% to 59%, averaging around
33%. The vast majority of false positives are ultimately shown to
be benign prostatic hyperplasia (BPH). The specificity is lowest
for modestly elevated tPSA, in the low so-called gray zone of 4 to
10 ng/ml. This low level of specificity results in additional more
invasive and costly diagnostic procedures, such as transrectal
ultrasounds and prostate biopsies. Such tests when unnecessary are
also very traumatic for the patient. The psychological impact of
being diagnosed as positive until proven as a false positive should
not be understated either.
[0007] Because of the shortcomings of tPSA, research has been
focused on attempting to develop PSA derivatives to increase the
sensitivity and specificity of this general diagnostic
approach.
[0008] One modification is free PSA (fPSA), which was FDA approved
in 1998. PSA in serum can be found either in an unbound form or
complexed with circulating protease inhibitors, most commonly with
alpha-1-antitrypsin (ACT). Clinicians have shown that the
proportion of PSA bound to ACT was significantly higher in men with
prostate cancer than in unaffected men or those with BPH. As a
guideline, if 25% orless of total PSA is free, this is an indicator
of possible prostate cancer. The fPSA assay was approved for use in
men with tPSA's for 4 to 10 ng/ml. Thus, the fPSA assay was
positioned to improve the specificity over that of tPSA alone.
However, the predictivity of the fPSA test is not as good in people
with really low or really high tPSA levels. Very low tPSA,
regardless of measured fPSA, is predictive of not having cancer,
while the converse is true with very high tPSA levels. The
diagnostic usefulness of fPSA is relatively limited as it can be
associated with either BPH or prostate cancer. The use of fPSA in
combination with tPSA has been shown to reduce the number of
unnecessary biopsies by about 20%.
[0009] Clearly, prostate biopsy is the gold standard for confirming
prostate cancer. However, even a biopsy is not always 100% certain.
The standard is the sextant biopsy where tissue sample collection
is guided by transrectal ultrasound. Often six samples are not
enough to detect the cancer and either a second biopsy procedure or
more than six samples are required.
[0010] Despite the improvements in prostate cancer screening over
the last ten years, there remains a large unmet need in diagnostic
sensitivity and specificity, even when these tools are used in
combination. Coupling this need with the large incidence of
prostate cancer and the importance for early, accurate detection,
the potential usefulness for a true differential diagnostic tool is
very significant.
[0011] A new prostate cancer marker, PCA3, was discovered a few
years ago by differential display analysis intended to highlight
genes associated with prostate cancer development. PCA3 is located
on chromosome 9 and composed of four exons. It encodes at least
four different transcripts which are generated by alternative
splicing and polyadenylation. By RT-PCR analysis, PCA3 expression
was found to be limited to the prostate and absent in all other
tissues tested, including testis, ovary, breast and bladder.
Northern blot analysis showed that PCA3 is highly expressed in the
vast majority of prostate cancers examined (47 out of 50) whereas
no or very low expression is detected in BPH or normal prostate
cells from the same patients [Cancer Res 1999 December
1;59(23):5975-9]. Moreover, a recent study comparing the clinical
performance of RNA telomerase RT and RNA PCA3 detection in the case
of prostate cancer showed that the PCA3 gene can be considered as a
better marker (Cancer Res 2002 May 1;62(9):2695-8).
[0012] The PCA3 gene is composed of 4 exons (e1-e4) and 3 introns
(i1-i3). While PCA3 appears to be recognized as the best
prostate-cancer marker ever identified, this specificity has been
contested in the literature. For example, Gandini et al. 2003,
claim that the prostate-specific expression of PCA3 is restricted
to that of exon 4 of the PCA3 gene. However, the applicants have
shown in a recent patent application that this is not the case
(Patent application CA 2,432,365).
[0013] In view of the fact that advanced prostate cancer remains a
life threatening disease reaching a very significant proportion of
the male population, there remains a need to provide the most
specific, selective, and rapid prostate cancer detection methods
and kits.
[0014] The present invention seeks to meet these and other
needs.
[0015] The present description refers to a number of documents, the
content of which is herein incorporated by reference in their
entirety.
SUMMARY OF THE INVENTION
[0016] The present invention relates to diagnostic methods and kits
to detect prostate cancer, which are more specific and selective
than the methods and kits of the prior art.
[0017] The present invention relates to a method to detect prostate
cancer in a patient and especially from a urine sample thereof by
detecting the RNA encoded by the PCA3 gene.
[0018] The invention further relates to a method of diagnosing the
presence or predisposition to develop prostate cancer in a patient.
Also disclosed is a method for monitoring the progression of
prostate cancer in a patient.
[0019] In one particular embodiment, the present invention relates
to a method to detect prostate cancer in urine samples by detecting
the presence of RNA encoded by the PCA3 gene. In one embodiment,
RNA encoded by the PCA3 gene is detected using an amplification
method, which simultaneously amplifies a second prostate-specific
nucleic acid sequence also contained in the sample.
[0020] In one further particular embodiment of the present
invention, the amplified second prostate specific marker is
selected from the group consisting of PSA, human kallikrein 2
(hK2/KLK2), prostate specific membrane antigen (PSMA),
transglutaminase 4, acid phosphatase or PCGEM1 RNA.
[0021] In another embodiment of the present invention, the RNA is
detected using an RNA amplification method. In a further
embodiment, the RNA amplification method is coupled to real-time
detection of the amplified products using fluorescence specific
probes. In yet a further embodiment, the amplification method is
PCR. In an additional embodiment the PCR is real-time PCR or a
related method enabling detection in real-time of the amplified
products.
[0022] In a related embodiment RNA encoded by the PCA3 gene is
detected in a nucleic acid extract by an in vitro RNA amplification
method named Nucleic Acid Based Amplification (NASBA). Of course
other RNA amplification methods are known and the instant methods
and kits are therefore not limited to NASBA. Non-limiting examples
of such RNA amplification methods include transcriptase mediated
amplification (TMA), strand displacement amplification (SDA) and
ligase chain reaction (LCR).
[0023] In a further embodiment, the amplified products are detected
in a homogenous phase using a fluorescent probe. In one embodiment,
the Beacon approach is used. In another embodiment, the product is
detected on solid phase using fluorescent or colorimetric method.
It should thus be understood that numerous fluorescent,
calorimetric or enzymatic methods can be used in accordance with
the present invention to detect and/or quantify RNAs. Other types
of labelled probes and primers or other types of detection methods
may also be used in the present invention (e.g., hybridization
assays such as Northern blots, dot blots or slot blots and
radiolabelled probes and primers).
[0024] In one embodiment, the RNA encoded by the PCA3 gene is
obtained from a cell contained in a voided urine sample from the
patient.
[0025] In another embodiment, the urine sample is obtained after an
attentive digital rectal examination (DRE). Of course, it should be
understood that the present methods and kits could also be used on
a urine sample obtained without DRE, or on other types of samples
such as sperm or mixed urine and sperm (e.g., first urine sample
following ejaculation), provided that the amplification method
and/or detection method is sensitive enough to detect the targeted
markers (PCA3 and second marker). Experiments showed that the
methods and kits of the present invention can also be performed
with these types of samples. Other samples that can be used include
blood or serum.
[0026] In one embodiment, the cells collected from the urine sample
are harvested and a total nucleic acid extraction is carried out.
In one particular embodiment, total nucleic acid extraction is
carried out using a solid phase band method on silica beads as
described by BOOM et al., (1990, J. Clin. Microbiol. 28: 495-503).
In another embodiment, the nucleic acids are purified using another
target capture method (see below). Of course, it should be
understood that numerous nucleic acid extraction and purification
methods exist and thus, that other methods could be used in
accordance with the present invention. Non-limiting examples
include a phenol/chloroform extraction method and target capture
purification method (see below). Other such methods are described
in herein referenced textbooks. It should also be recognized that
numerous means to stabilize or protect the prostate cells contained
in the urine sample or other sample, as well as to stabilize or
protect the RNA present in these cells are well known in the
art.
[0027] In another embodiment, the methods of the present invention
are carried out using a crude, unpurified, or semi-purified
sample.
[0028] In one particular embodiment, the present invention also
relates to a prostate cancer diagnostic kit for detecting the
presence of PCA3 nucleic acid in a sample. Such kit generally
comprises a first container means having disposed therein at least
one oligonucleotide probe and/or primer that hybridizes to a PCA3
nucleic acid (e.g. PCA3 RNA) and a second container means
containing at least one other oligonucleotide primer and/or probe
that hybridizes to the above-mentioned second prostate-specific
sequence. In another embodiment, a third container means contains a
probe which specifically hybridizes to the PCA3 amplification
product. In a preferred embodiment, the kit further includes other
containers comprising additional components such as a additional
oligonucleotide or primer and/or one or more of the following:
buffers, reagents to be used in the assay (e.g. wash reagents,
polymerases, internal controls (IC) or else) and reagents capable
of detecting the presence of bound nucleic acid probe(s)/primer(s).
Of course numerous embodiments of the kits of the present invention
are possible. For example, the different container means can be
divided in amplifying reagents and detection reagents. In one such
an embodiment, a first container means contains amplification or
hybridization reagents specific for the target nucleic acids of the
present invention (e.g., PCA 3, second prostate specific and
internal control nucleic acids) and the second container means
contains detection reagents. Alternatively, the detection reagents
and amplification reagents can be contained in the same container
mean.
[0029] The present invention in addition relates to a prostate
cancer diagnostic kit for detecting the presence of PCA3 nucleic
acid in a sample. Such kit generally comprises a first container
means having disposed therein at least one oligonucleotide probe
and/or primer that hybridizes to a PCA3 mRNA and a second container
means containing at least one other oligonucleotide primer and/or
probe that hybridizes to the mRNA of the second prostate-specific
sequence. In another embodiment, a third container means contains a
probe which specifically hybridizes to the PCA3 amplification
product. In a yet another embodiment a fourth container means
contains a probe which specifically hybridizes to the second
prostate specific mRNA. In a preferred embodiment, the kit further
includes other containers comprising additional components such as
a additional oligonucleotide or primer (e.g., for internal control)
and/or one or more of the following: buffers, reagents to be used
in the assay (e.g. wash reagents, polymerases, internal control
nucleic acid or cells or else) and reagents capable of detecting
the presence of bound nucleic acid probe(s)/primer(s). Of course
the separation or assembly of reagents in same or different
container means is dictated by the types of extraction,
amplification or hybridization methods, and detection methods used
as well as other parameters including stability, need for
preservation etc.
[0030] Multiple methods and kits are encompassed by the present
invention. For example, the detection and or amplification of the
PCA3 nucleic acid sequence does not need to be identical to that of
the second prostate specific polynucleotide or other targeted
sequences. Thus for example a method or kit which would be RNA
based for PCA3 could be DNA based for the second prostate marker or
for other targeted sequences.
[0031] It should be understood by a person of ordinary skill that
numerous statistical methods can be used in the context of the
present invention to determine if the test is positive or negative.
The decisional tree used is only one non-limiting example of such a
statistical method.
[0032] Unless defined otherwise, the scientific and technological
terms and nomenclature used herein have the same meaning as
commonly understood by a person of ordinary skill to which this
invention pertains. Commonly understood definitions of molecular
biology terms can be found for example in Dictionary of
Microbiology and Molecular Biology, 2nd ed. (Singleton et al.,
1994, John Wiley & Sons, New York, N.Y.) or The Harper Collins
Dictionary of Biology (Hale & Marham, 1991, Harper Perennial,
New York, N.Y.), Rieger et al., Glossary of genetics: Classical and
molecular, 5.sup.th edition, Springer-Verlag, N.Y., 1991; Alberts
et al., Molecular Biology of the Cell, 4.sup.th edition, Garland
science, New-York, 2002; and, Lewin, Genes VII, Oxford University
Press, New-York, 2000. Generally, the procedures of molecular
biology methods and the like are common methods used in the art.
Such standard techniques can be found in reference manuals such as
for example Sambrook et al. (2000, Molecular Cloning--A Laboratory
Manual, Third Edition, Cold Spring Harbor Laboratories); and
Ausubel et al. (1994, Current Protocols in Molecular Biology, John
Wiley & Sons, New-York).
[0033] In the present description, a number of terms are
extensively utilized. In order to provide a clear and consistent
understanding of the specification and claims, including the scope
to be given such terms, the following definitions are provided.
DEFINITIONS
[0034] Nucleotide sequences are presented herein by single strand,
in the 5' to 3' direction, from left to right, using the one letter
nucleotide symbols as commonly used in the art and in accordance
with the recommendations of the IUPAC-IUB Biochemical Nomenclature
Commission.
[0035] The present description refers to a number of routinely used
recombinant DNA (rDNA) technology terms. Nevertheless, definitions
of selected examples of such rDNA terms are provided for clarity
and consistency.
[0036] As used herein, "nucleic acid molecule" or
"polynucleotides", refers to a polymer of nucleotides. Non-limiting
examples thereof include DNA (e.g. genomic DNA, cDNA), RNA
molecules (e.g. mRNA) and chimeras thereof. The nucleic acid
molecule can be obtained by cloning techniques or synthesized. DNA
can be double-stranded or single-stranded (coding strand or
non-coding strand [antisense]). Conventional ribonucleic acid (RNA)
and deoxyribonucleic acid (DNA) are included in the term "nucleic
acid" and polynucleotides as are analogs thereof. A nucleic acid
backbone may comprise a variety of linkages known in the art,
including one or more of sugar-phosphodiester linkages,
peptide-nucleic acid bonds (referred to as "peptide nucleic acids"
(PNA); Hydig-Hielsen et al., PCT Int'l Pub. No. WO 95/32305),
phosphorothioate linkages, methylphosphonate linkages or
combinations thereof. Sugar moieties of the nucleic acid may be
ribose or deoxyribose, or similar compounds having known
substitutions, e.g., 2' methoxy substitutions (containing a
2'-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2'
halide substitutions. Nitrogenous bases may be conventional bases
(A, G, C, T, U), known analogs thereof (e.g., inosine or others;
see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed.,
11.sup.th ed., 1992), or known derivatives of purine or pyrimidine
bases (see, Cook, PCT Int'l Pub. No. WO 93/13121) or "abasic"
residues in which the backbone includes no nitrogenous base for one
or more residues (Arnold et al., U.S. Pat. No. 5,585,481). A
nucleic acid may comprise only conventional sugars, bases and
linkages, as found in RNA and DNA, or may include both conventional
components and substitutions (e.g., conventional bases linked via a
methoxy backbone, or a nucleic acid including conventional bases
and one or more base analogs). The terminology "PCA3 nucleic acid"
or "PCA3 polynucleotides" refers to a native PCA3 nucleic acid
sequence. In one embodiment, the PCA3 nucleic acid has the sequence
has set forth in SEQ ID NOs 9,10 and 13. In another embodiment, the
PCA3 nucleic acid encodes a PCA3 protein. In a further embodiment,
the PCA3 nucleic acid is a non-coding nucleic acid sequence. In yet
a further embodiment, the PCA3 sequence which is targeted by the
PCA3 sequences encompassed by the present invention, is a natural
PCA3 sequence found in a patient sample.
[0037] The term "recombinant DNA" as known in the art refers to a
DNA molecule resulting from the joining of DNA segments. This is
often referred to as genetic engineering. The same is true for
"recombinant nucleic acid".
[0038] The term "DNA segment" is used herein, to refer to a DNA
molecule comprising a linear stretch or sequence of nucleotides.
This sequence when read in accordance with the genetic code (e.g.,
an open reading frame or ORF), can encode a linear stretch or
sequence of amino acids which can be referred to as a polypeptide,
protein, protein fragment and the like.
[0039] The terminology "amplification pair" or "primer pair" refers
herein to a pair of oligonucleotides (oligos) of the present
invention, which are selected to be used together in amplifying a
selected nucleic acid sequence by one of a number of types of
amplification processes.
[0040] "Amplification" refers to any known in vitro procedure for
obtaining multiple copies ("amplicons") of a target nucleic acid
sequence or its complement or fragments thereof. In vitro
amplification refers to production of an amplified nucleic acid
that may contain less than the complete target region sequence or
its complement. Known in vitro amplification methods include, e.g.,
transcription-mediated amplification, replicase-mediated
amplification, polymerase chain reaction (PCR) amplification,
ligase chain reaction (LCR) amplification and strand-displacement
amplification (SDA). Replicase-mediated amplification uses
self-replicating RNA molecules, and a replicase such as
Q.beta.-replicase (e.g., Kramer et al., U.S. Pat. No. 4,786,600).
PCR amplification is well known and uses DNA polymerase, primers
and thermal cycling to synthesize multiple copies of the two
complementary strands of DNA or cDNA (e.g., Mullis et al., U.S.
Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification
uses at least four separate oligonucleotides to amplify a target
and its complementary strand by using multiple cycles of
hybridization, ligation, and denaturation (e.g., EP Pat. App. Pub.
No.0 320 308). SDA is a method in which a primer contains a
recognition site for a restriction endonuclease that permits the
endonuclease to nick one strand of a hemimodified DNA duplex that
includes the target sequence, followed by amplification in a series
of primer extension and strand displacement steps (e.g., Walker et
al., U.S. Pat. No. 5,422,252). Another known strand-displacement
amplification method does not require endonuclease nicking
(Dattagupta et al., U.S. Pat. No. 6,087,133).
Transcription-mediated amplification is used in the present
invention. Those skilled in the art will understand that the
oligonucleotide primer sequences of the present invention may be
readily used in any in vitro amplification method based on primer
extension by a polymerase. (see generally Kwoh et al., 1990, Am.
Biotechnol. Lab. 8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad.
Sci. USA 86, 1173-1177; Lizardi et al., 1988, BioTechnology
6:1197-1202; Malek et al., 1994, Methods Mol. Biol., 28:253-260;
and Sambrook et al., 2000, Molecular Cloning--A Laboratory Manual,
Third Edition, CSH Laboratories). As commonly known in the art, the
oligos are designed to bind to a complementary sequence under
selected conditions.
[0041] As used herein, the term "physiologically relevant" is meant
to describe interactions that can modulate a function which is
physiologically relevant. In the present invention, encompassed for
example the transcription of a gene in its natural setting. Of
course a binding of a protein to PCA3 may also be considered as a
physiologically relevant function if this binding occur in a
natural setting.
[0042] The term "DNA" molecule or sequence (as well as sometimes
the term "oligonucleotide") refers to a molecule comprised
generally of the deoxyribonucleotides adenine (A), guanine (G),
thymine (T) and/or cytosine (C). In "RNA", T is replaced by uracil
(U). As used herein, particular DNA or RNA sequences may be
described according to the normal convention of giving only the
sequence in the 5' to 3' direction.
[0043] Agarose Gel Electrophoresis. The most commonly used
technique (though not the only one) for fractionating double
stranded DNA is agarose gel electrophoresis. The principle of this
method is that DNA molecules migrate through the gel as though it
were a sieve that retards the movement of the largest molecules to
the greatest extent and the movement of the smallest molecules to
the least extent. Note that the smaller the DNA fragment, the
greater the mobility under electrophoresis in the agarose gel.
[0044] The DNA fragments fractionated by agarose gel
electrophoresis can be visualized directly by a staining procedure
if the numberof fragments included in the pattern is small. In
ordertovisualize a small subset of these fragments, a methodology
referred to as a hybridization procedure (e.g., Southern
hybridization) can be applied.
[0045] "Nucleic acid hybridization" refers generally to the
hybridization of two single-stranded nucleic acid molecules having
complementary base sequences, which under appropriate conditions
will form a thermodynamically favored double-stranded structure.
Examples of hybridization conditions can be found in the two
laboratory manuals referred above (Sambrook et al., 2000, supra and
Ausubel et al., 1994, supra) and are commonly known in the art. In
the case of a hybridization to a nitrocellulose filter (or other
such support like nylon), as for example in the well known Southern
blotting procedure, a nitrocellulose filter can be incubated
overnight at 65.degree. C. with a labeled probe in a solution
containing high salt (6.times.SSC or 5.times.SSPE), 5.times.
Denhardt's solution, 0.5% SDS, and 100 .mu.g/ml denatured carrier
DNA (e.g. salmon sperm DNA). The non-specifically binding probe can
then be washed off the filter by several washes in
0.2.times.SSC/0.1% SDS at a temperature which is selected in view
of the desired stringency: room temperature (low stringency),
42.degree. C. (moderate stringency) or 65.degree. C. (high
stringency). The salt and SDS concentration of the washing
solutions may also be adjusted to accommodate for the desired
stringency. The selected temperature and salt concentration is
based on the melting temperature (Tm) of the DNA hybrid. Of course,
RNA-DNA hybrids can also be formed and detected. In such cases, the
conditions of hybridization and washing can be adapted according to
well known methods by the person of ordinary skill. Stringent
conditions will be preferably used (Sambrook et al., 2000, supra).
Other protocols or commercially available hybridization kits (e.g.,
ExpressHyb.TM. from BD Biosciences Clonetech) using different
annealing and washing solutions can also be used as well known in
the art.
[0046] A "probe" is meant to include a nucleic acid oligomer that
hybridizes specifically to a target sequence in a nucleic acid or
its complement, under conditions that promote hybridization,
thereby allowing detection of the target sequence or its amplified
nucleic acid. Detection may either be direct (i.e, resulting from a
probe hybridizing directly to the target or amplified sequence) or
indirect (i.e., resulting from a probe hybridizing to an
intermediate molecular structure that links the probe to the target
or amplified sequence). A probe's "target" generally refers to a
sequence within an amplified nucleic acid sequence (i.e, a subset
of the amplified sequence) that hybridizes specifically to at least
a portion of the probe sequence by standard hydrogen bonding or
"base pairing." Sequences that are "sufficiently complementary"
allow stable hybridization of a probe sequence to a target
sequence, even if the two sequences are not completely
complementary. A probe may be labeled or unlabeled.
[0047] By "sufficiently complementary" is meant a contiguous
nucleic acid base sequence that is capable of hybridizing to
another sequence by hydrogen bonding between a series of
complementary bases. Complementary base sequences may be
complementary at each position in sequence by using standard base
pairing (e.g., G:C, A:T or A:U pairing) or may contain one or more
residues (including abasic residues) that are not complementary by
using standard base pairing, but which allow the entire sequence to
specifically hybridize with another base sequence in appropriate
hybridization conditions. Contiguous bases of an oligomer are
preferably at least about 80% (81, 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%), more preferably at
least about 90% complementary to the sequence to which the oligomer
specifically hybridizes. Appropriate hybridization conditions are
well known to those skilled in the art, can be predicted readily
based on sequence composition and conditions, or can be determined
empirically by using routine testing (see Sambrook et al.,
Molecular Cloning, A Laboratoiy Manual, 2.sup.nd ed. (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) at
.sctn..sctn. 1.90-1.91, 7.37-7.57, 9.47-9.51 and 11.47-11.57,
particularly at .sctn..sctn. 9.50-9.51, 11.12-11.13, 11.45-11.47
and 11.55-11.57).
[0048] Nucleic acid sequences may be detected by using
hybridization with a complementary sequence (e.g., oligonucleotide
probes) (see U.S. Pat. No. 5,503,980 (Cantor), U.S. Pat. No.
5,202,231 (Drmanac et al.), U.S. Pat. No. 5,149,625 (Church et
al.), U.S. Pat. No. 5,112,736 (Caldwell et al.), U.S. Pat. No.
5,068,176 (Vijg et al.), and U.S. Pat. No. 5,002,867 (Macevicz)).
Hybridization detection methods may use an array of probes (e.g.,
on a DNA chip) to provide sequence information about the target
nucleic acid which selectively hybridizes to an exactly
complementary probe sequence in a set of four related probe
sequences that differ one nucleotide (see U.S. Pat. Nos. 5,837,832
and 5,861,242 (Chee et al.)).
[0049] A detection step may use any of a variety of known methods
to detect the presence of nucleic acid by hybridization to a probe
oligonucleotide. One specific example of a detection step uses a
homogeneous detection method such as described in detail previously
in Arnold et al. Clinical Chemistry 35:1588-1594 (1989), and U.S.
Pat. No. 5,658,737 (Nelson et al.), and U.S. Pat. Nos. 5,118,801
and 5,312,728 (Lizardi et al.).
[0050] The types of detection methods in which probes can be used
include Southern blots (DNA detection), dot or slot blots (DNA,
RNA), and Northern blots (RNA detection). Labeled proteins could
also be used to detect a particular nucleic acid sequence to which
it binds (e.g protein detection by far western technology: Guichet
et al., 1997, Nature 385(6616): 548-552; and Schwartz et al., 2001,
EMBO 20(3): 510-519). Other detection methods include kits
containing reagents of the present invention on a dipstick setup
and the like. Of course, it might be preferable to use a detection
method which is amenable to automation. A non-limiting example
thereof includes a chip or other support comprising one or more
(e.g. an array) of different probes.
[0051] A "label" refers to a molecular moiety or compound that can
be detected or can lead to a detectable signal. A label is joined,
directly or indirectly, to a nucleic acid probe or the nucleic acid
to be detected (e.g., an amplified sequence). Direct labeling can
occur through bonds or interactions that link the label to the
nucleic acid (e.g., covalent bonds or non-covalent interactions),
whereas indirect labeling can occur through use a "linker" or
bridging moiety, such as additional oligonucleotide(s), which is
either directly or indirectly labeled. Bridging moieties may
amplify a detectable signal. Labels can include any detectable
moiety (e.g., a radionuclide, ligand such as biotin or avidin,
enzyme or enzyme substrate, reactive group, chromophore such as a
dye or colored particle, luminescent compound including a
bioluminescent, phosphorescent or chemiluminescent compound, and
fluorescent compound). Preferably, the label on a labeled probe is
detectable in a homogeneous assay system, i.e., in a mixture, the
bound label exhibits a detectable change compared to an unbound
label.
[0052] Other methods of labeling nucleic acids are known whereby a
label is attached to a nucleic acid strand as it is fragmented,
which is useful for labeling nucleic acids to be detected by
hybridization to an array of immobilized DNA probes (e.g., see PCT
No. PCT/IB99/02073).
[0053] A "homogeneous detectable label" refers to a label whose
presence can be detected in a homogeneous fashion based upon
whether the labeled probe is hybridized to a target sequence. A
homogeneous detectable label can be detected without physically
removing hybridized from unhybridized forms of the labeled probe.
Homogeneous detectable labels and methods of detecting them have
been described in detail elsewhere (e.g., see U.S. Pat. Nos.
5,283,174, 5,656,207 and 5,658,737).
[0054] As used herein, "oligonucleotides" or "oligos" define a
molecule having two or more nucleotides (ribo or
deoxyribonucleotides). The size of the oligo will be dictated by
the particular situation and ultimately on the particular use
thereof and adapted accordingly by the person of ordinary skill. An
oligonucleotide can be synthesized chemically or derived by cloning
according to well known methods. While they are usually in a
single-stranded form, they can be in a double-stranded form and
even contain a "regulatory region". They can contain natural rare
or synthetic nucleotides. They can be designed to enhance a chosen
criteria like stability for example.
[0055] As used herein, a "primer" defines an oligonucleotide which
is capable of annealing to a target sequence, thereby creating a
double stranded region which can serve as an initiation point for
nucleic acid synthesis under suitable conditions. Primers can be,
for example, designed to be specific for certain alleles so as to
be used in an allele-specific amplification system. For example, a
primer can be designed so as to be complementary to a short PCA3
RNA which is associated with a malignant state of the prostate,
whereas a long PCA3 RNA is associated with a non-malignant state
(benign) thereof (PCT/CA00/01154 published under No. WO 01/23550).
The primer's 5' region may be non-complementary to the target
nucleic acid sequence and include additional bases, such as a
promoter sequence (which is referred to as a "promoter primer").
Those skilled in the art will appreciate that any oligomer that can
function as a primer can be modified to include a 5' promoter
sequence, and thus function as a promoter primer. Similarly, any
promoter primer can serve as a primer, independent of its
functional promoter sequence. Of course the design of a primer from
a known nucleic acid sequence is well known in the art. As for the
oligos, it can comprise a number of types of different
nucleotides.
[0056] Transcription-associated amplification. Amplifying a target
nucleic acid sequence by using at least two primers can be
accomplished using a variety of known nucleic acid amplification
methods, but preferably uses a transcription-associated
amplification reaction that is substantially isothermal. By using
such an in vitro amplification method, many strands of nucleic acid
are produced from a single copy of target nucleic acid, thus
permitting detection of the target in the sample by specifically
binding the amplified sequences to one or more detection probes.
Transcription-associated amplification methods have been described
in detail elsewhere (e.g., U.S. Pat. Nos. 5,399,491 and 5,554,516).
Briefly, transcription-associated amplification uses two types of
primers (one being a promoter primer because it contains a promoter
sequence for an RNA polymerase), two enzyme activities (a reverse
transcriptase (RT) and an RNA polymerase), substrates
(deoxyribonucleoside triphosphates, ribonucleoside triphosphates)
and appropriate salts and buffers in solution to produce multiple
RNA transcripts from a nucleic acid template. Initially, a promoter
primer hybridizes specifically to a target sequence (e.g., RNA) and
reverse transcriptase creates a first complementary DNA strand
(cDNA) by extension from the 3' end of the promoter primer. The
cDNA is made available for hybridization with the second primer by
any of a variety of methods, such as, by denaturing the target-cDNA
duplex or using RNase H activity supplied by the RT that degrades
RNA in a DNA:RNA duplex. A second primer binds to the cDNA and a
new strand of DNA is synthesized from the end of the second primer
using the RT activity to create a double-stranded DNA (dsDNA)
having a functional promoter sequence at one end. An RNA polymerase
binds to the dsDNA promoter sequence and transcription produces
multiple transcripts ("amplicons"). Amplicons are used in
subsequent steps or cycles of the transcription-associated
amplification process by serving as a new template for replication,
thus generating many copies of amplified nucleic acid (i.e., about
100 to 3,000 copies of RNA are synthesized from each template).
[0057] NASBA. Nucleic Acid Sequence Based Amplification (NASBA) can
be carried out in accordance with known techniques (Malek et al.
Methods Mol Biol, 28:253-260). In an embodiment, the NASBA
amplification starts with the annealing of an antisense primer P1
(containing the T7 RNA polymerase promoter) to the mRNA target.
Reverse transcriptase (RTase) then synthesizes a complementary DNA
strand. The double stranded DNA/RNA hybrid is recognized by RNase H
that digests the RNA strand, leaving a single-stranded DNA molecule
to which the sense primer P2 can bind. P2 serves as an anchor to
the RTase that synthesizes a second DNA strand. The resulting
double-stranded DNA has a functional T7 RNA polymerase promoter
recognized by the respective enzyme. The NASBA reaction can then
enter in the phase of cyclic amplification comprising six steps:
(1) Synthesis of short antisense single-stranded RNA molecules
(10.sup.1 to 10.sup.3 copies per DNA template) by the T7 RNA
polymerase; (2) annealing of primer P2 to these RNA molecules; (3)
synthesis of a complementary DNA strand by RTase; (4) digestion of
the RNA strand in the DNA/RNA hybrid; (5) annealing of primer P1 to
the single-stranded DNA; and (6) generation of double stranded DNA
molecules by RTase. Because the NASBA reaction is isothermal
(41.degree. C.), specific amplification of ssRNA is possible if
denaturation of dsDNA is prevented in the sample preparation
procedure. It is thus possible to pick up RNA in a dsDNA background
without getting false positive results caused by genomic dsDNA.
[0058] Polymerase chain reaction (PCR). Polymerase chain reaction
can be carried out in accordance with known techniques. See, e.g.,
U.S. Pat. Nos. 4,683,195; 4,683,202; 4,800,159; and 4,965,188 (the
disclosures of all three U.S. Patent are incorporated herein by
reference). In general, PCR involves, a treatment of a nucleic acid
sample (e.g., in the presence of a heat stable DNA polymerase)
under hybridizing conditions, with one oligonucleotide primer for
each strand of the specific sequence to be detected. An extension
product of each primer which is synthesized is complementary to
each of the two nucleic acid strands, with the primers sufficiently
complementary to each strand of the specific sequence to hybridize
therewith. The extension product synthesized from each primer can
also serve as a template for further synthesis of extension
products using the same primers. Following a sufficient number of
rounds of synthesis of extension products, the sample is analyzed
to assess whether the sequence or sequences to be detected are
present. Detection of the amplified sequence may be carried out by
visualization following EtBr staining of the DNA following gel
electrophoresis, or using a detectable label in accordance with
known techniques, and the like. For a review on PCR techniques (see
PCR Protocols, A Guide to Methods and Amplifications, Michael et
al. Eds, Acad. Press, 1990).
[0059] Ligase chain reaction (LCR) can be carried out in accordance
with known techniques (Weiss, 1991, Science 254:1292). Adaptation
of the protocol to meet the desired needs can be carried out by a
person of ordinary skill. Strand displacement amplification (SDA)
is also carried out in accordance with known techniques or
adaptations thereof to meet the particular needs (Walker et al.,
1992, Proc. Natl. Acad. Sci. USA 89:392-396; and ibid., 1992,
Nucleic Acids Res. 20:1691-1696).
[0060] Target capture. In one embodiment, target capture is
included in the method to increase the concentration or purity of
the target nucleic acid before in vitro amplification. Preferably,
target capture involves a relatively simple method of hybridizing
and isolating the target nucleic acid, as described in detail
elsewhere (e.g., see U.S. Pat. Nos. 6,110,678, 6,280,952, and
6,534,273). Generally speaking, target capture can be divided in
two family, sequence specific and non sequence specific. In the
non-specific method, a reagent (e.g., silica beads) is used to
capture non specifically nucleic acids. In the sequence specific
method an oligonucleotide attached to a solid support is contacted
with a mixture containing the target nucleic acid under appropriate
hybridization conditions to allow the target nucleic acid to be
attached to the solid support to allow purification of the target
from other sample components. Target capture may result from direct
hybridization between the target nucleic acid and an
oligonucleotide attached to the solid support, but preferably
results from indirect hybridization with an oligonucleotide that
forms a hybridization complex that links the target nucleic acid to
the oligonucleotide on the solid support. The solid support is
preferably a particle that can be separated from the solution, more
preferably a paramagnetic particle that can be retrieved by
applying a magnetic field to the vessel. After separation, the
target nucleic acid linked to the solid support is washed and
amplified when the target sequence is contacted with appropriate
primers, substrates and enzymes in an in vitro amplification
reaction.
[0061] Generally, capture oligomer sequences include a sequence
that specifically binds to the target sequence, when the capture
method is indeed specific, and a "tail" sequence that links the
complex to an immobilized sequence by hybridization. That is, the
capture oligomer includes a sequence that binds specifically to its
PCA3 or to another prostate specific marker (e.g., PSA, hK2/KLK2,
PMSA, transglutaminase 4, acid phosphatase, PCGEM1) target sequence
and a covalently attached 3' tail sequence (e.g., a homopolymer
complementary to an immobilized homopolymer sequence). The tail
sequence which is, for example, 5 to 50 nucleotides long,
hybridizes to the immobilized sequence to link the
target-containing complex to the solid support and thus purify the
hybridized target nucleic acid from other sample components. A
capture oligomer may use any backbone linkage, but some embodiments
include one or more 2'-methoxy linkages. Of course, other capture
methods are well known in the art. The capture method on the cap
structure (Edery et al., 1988, gene 74(2): 517-525, U.S. Pat. No.
5,219,989) or the silica based method are two non-limiting examples
of capture methods.
[0062] An "immobilized probe" or "immobilized nucleic acid" refers
to a nucleic acid that joins, directly or indirectly, a capture
oligomer to a solid support. An immobilized probe is an oligomer
joined to a solid support that facilitates separation of bound
target sequence from unbound material in a sample. Any known solid
support may be used, such as matrices and particles free in
solution, made of any known material (e.g., nitrocellulose, nylon,
glass, polyacrylate, mixed polymers, polystyrene, silane
polypropylene and metal particles, preferably paramagnetic
particles). Preferred supports are monodisperse paramagnetic
spheres (i.e., uniform in size.+-.about 5%), thereby providing
consistent results, to which an immobilized probe is stably joined
directly (e.g., via a direct covalent linkage, chelation, or ionic
interaction), or indirectly (e.g., via one or more linkers),
permitting hybridization to another nucleic acid in solution.
[0063] The term "allele" defines an alternative form of a gene
which occupies a given locus on a chromosome.
[0064] Gene. A DNA sequence generally related but not necessarely
related to a single polypeptide chain or protein, and as used
herein includes the 5' and 3' untranslated regions. The polypeptide
can be encoded by a full-length sequence or any portion of the
coding sequence, so long as the functional activity of the protein
is retained.
[0065] Complementary DNA (cDNA). Recombinant nucleic acid molecules
synthesized by reverse transcription of messenger RNA ("RNA").
[0066] Structural Gene. A DNA sequence that is transcribed into RNA
that is then translated into a sequence of amino acids
characteristic of a specific polypeptide(s).
[0067] As commonly known, a "mutation" is a detectable change in
the genetic material which can be transmitted to a daughter cell.
As well known, a mutation can be, for example, a detectable change
in one or more deoxyribonucleotide. For example, nucleotides can be
added, deleted, substituted for, inverted, or transposed to a new
position. Spontaneous mutations and experimentally induced
mutations exist. A mutant polypeptide can be encoded from this
mutant nucleic acid molecule.
[0068] As used herein, the term "purified" refers to a molecule
(e.g. nucleic acid) having been separated from a component of the
composition in which it was originally present. Thus, for example,
a "purified nucleic acid" has been purified to a level not found in
nature. A "substantially pure" molecule is a molecule that is
lacking in most other components (e.g., 30, 40, 50, 60, 70, 75, 80,
85, 90, 95, 96, 97, 98, 99, 100% free of contaminants). By
opposition, the term "crude" means molecules that have not been
separated from the components of the original composition in which
it was present. For the sake of brevity, the units (e.g. 66, 67 . .
. 81, 82, . . . 91, 92% . . . ) have not been specifically recited
but are considered nevertheless within the scope of the present
invention.
[0069] As used herein the terminology "prostate specific marker"
relates to any molecule whose presence in the sample indicates that
such sample contains prostate cells (or a marker therefrom).
Therefore a "prostate specific sequence" refers to a nucleic acid
or protein sequence specifically found in prostate cells and
usually not in other tissues which could "contaminate" a particular
sample. For certainty, when a urine sample is used, the second
prostate specific marker according to the present invention does
not have to be solely expressed in the prostate. In fact markers
which are solely expressed in one organ or tissue is very rare.
However, should the second prostate specific marker be expressed in
non-prostate tissue, this non prostate tissue expression will not
jeopardized the specificity of this second marker provided that it
occurs in cells of tissues or organs which are not normally present
in the urine sample. Thus, when urine is the sample, this second
prostate-specific marker is not normally expressed in other types
of cells (e.g., cells from the urinary tract system) to be found in
the urine sample.
[0070] Control sample. By the term "control sample" or "normal
sample" is meant here a sample that does not contain a specifically
chosen cancer. In a particular embodiment, the control sample does
not contain prostate cancer or is indicative of the absence of
prostate cancer. Control samples can be obtained from
patients/individuals not afflicted with prostate cancer. Other
types of control samples may also be used. For example, a prostate
specific marker can be used as to make sure that the sample
contains prostate specific cells (this marker is generally
described herein as the second prostate-specific marker). In a
related aspect, a control reaction may be designed to control the
method itself (e.g., The cell extraction, the capture, the
amplification reaction or detection method, number of cells present
in the sample, a combination thereof or any step which could be
monitored to positively validate that the absence of a signal
(e.g., the absence of PCA3 signal) is not the result of a defect in
one ore more of the steps).
[0071] Cut-off value. The cut-off value for the predisposition or
presence of prostate cancer is defined from a population of
patients without prostate cancer as the average signal of PCA3 (or
other prostate cancer antigen) polynucleotides, polypeptides or
fragments thereof plus n standard deviations (or average mean
signal thereof). Cut off values indicative of the presence or
predisposition to develop prostate cancer may be the same or
alternatively, they may be different values.
[0072] Variant. The term "variant" refers herein to a protein or
nucleic acid molecule which is substantially similar in structure
and biological activity to the protein or nucleic acid of the
present invention, to maintain at least one of its biological
activities. Thus, provided that two molecules possess a common
activity and can substitute for each other, they are considered
variants as that term is used herein even if the composition, or
secondary, tertiary or quaternary structure of one molecule is not
identical to that found in the other, or if the amino acid sequence
or nucleotide sequence is not identical.
[0073] A "biological sample" or "sample of a patient" is meant to
include any tissue or material derived from a living or dead human
which may contain the PCA3 target nucleic acid and second prostate
specific marker. Samples include, for example, any tissue or
material that may contain cells specific for the PCA3 target (or
second specific marker), such as peripheral blood, plasma or serum,
biopsy tissue, gastrointestinal tissue, bone marrow, urine, feces,
semen or other body fluids, tissues or materials, but preferably is
a urine sample following digital rectal examination (or other means
which increase the content of prostate cells in urine). The
biological sample may be treated to physically disrupt tissue or
cell structure, thus releasing intracellular components into a
solution which may further contain enzymes, buffers, salts,
detergents, and the like which are used to prepare the sample for
analysis.
[0074] Other objects, advantages and features of the present
invention will become more apparent upon reading of the following
non-restrictive description of illustrative embodiments thereof,
given by way of example only with reference to the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0075] Having thus, generally described the invention, reference
will be made to the accompanying drawings, showing by way of
illustration only an illustrative embodiment thereof and in
which:
[0076] FIG. 1 shows the PCA3 gene structure and location of
oligonucleotides and probes for in vitro RNA amplification and
amplified product detection. In accordance with one embodiment of
the present invention. Panel A. Targeting zone of sense PCA3 primer
(SEQ ID NO 4); Panel B. Targeting zone of PCA3 molecular beacon
(SEQ ID NO 6); and Panel C. Targeting zone of anti-sense PCA3
primer (SEQ ID NO 3).
[0077] FIG. 2 shows a decisional tree used to calculate the
positivity of the method in a patient with total blood PSA below 4
ng/ml.
[0078] FIG. 3 shows a decisional tree used to calculate the
positivity of the method in a patient with total blood PSA between
4-10 ng/ml.
[0079] FIG. 4 shows a decisional tree used to calculate the
positivity of the method in a patient with total blood PSA above 10
ng/ml.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0080] For purposes of clarity of disclosure, and not by way of
limitation, the detailed description of the invention is divided
into the following subsections:
[0081] I. A Method to Assess the Presence of Prosate Cancer in a
Sample by Detecting PCA3 Nucleic Acid.
[0082] II. Synthesis of Nucleic Acid.
[0083] III. Probes and Primers.
[0084] IV. A Kit for Detecting the Presence of PCA3 Nucleic Acid in
a Sample.
[0085] 1. A Method to Assess the Presence of Prosate Cancer in a
Sample by Detecting PCA3 nucleic acid
[0086] The invention encompasses methods for detecting the presence
of a PCA3 nucleic acid together with a second prostate specific
marker (e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid
phophatase, PCGEM1) in a biological sample as well as methods for
measuring the level of a PCA3 nucleic acid in the sample. Such
methods are useful for the diagnostic of prostate cancers
associated with PCA3 overexpression.
[0087] The predisposition to develop prostate cancer or the
presence of such cancer may be detected based on the presence of an
elevated amount of PCA3 nucleic acid in a biological sample (e.g.,
urine) of a patient. Polynucleotides primers and probes may be used
to detect the level of PCA3 RNA present, which is indicative of the
predisposition, presence or absence of prostate cancer. In general
the elevated amount of PCA3 nucleic acid (e.g., PCA3 mRNA or
fragments thereof) in a sample as compared to the amount present in
a normal control samples (or a determined cut-off value) indicates
that the sample contains prostate cancer or is susceptible to
develop prostate cancer. In one embodiment, the detection of a
second prostate-specific marker is also performed to serve as a
control for the presence of prostate specific cells in the sample
as well as to further validate the PCA3 detection results (e.g., a
negative result obtained with the detection of PCA3).
[0088] Of course, a number of different prostate specific marker
can be used as long as they can serve as a control for prostate
RNA. Non-limiting examples of such prostate-specific markers
include PSA (SEQ ID NO 11) and other Kallikrein family members. In
addition and as described above, markers such as hK2/KLK2, PSMA,
transglutaminase 4, acid phosphatase, PCGEM1 can also be used in
accordance with the present invention.
[0089] One non limiting example of a method to detect PCA3 nucleic
acid (e.g. PCA3 mRNA) in a biological sample is by (1) contacting a
biological sample with at least one oligonucleotide probe or primer
that hybridizes to a PCA3 polynucleotide; and (2) detecting in the
biological sample a level of oligonucleotide (i.e. probe(s) or
primer(s)) that hybridizes to the PCA3 polynucleotide. The sample
is also tested for the presence of second prostate-specific marker
(e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase,
PCGEM1 mRNA or fragments thereof) to control for the presence of
prostate cells in the sample (or their number) as well as to
further control a negative or positive result obtained with the
detection of PCA3. The second prostate specific marker may also be
a prostate specific PCA3 RNA that is not associated with prostate
cancer but is expressed in prostate cells. The amount of PCA3
polynucleotide detected can be compared with a predetermined cut
off value, and therefrom the predisposition, presence or absence of
a prostate cancer in the patient is determined.
[0090] In a related aspect, the methods of the present invention
can be used for monitoring the progression of prostate cancer in a
patient. In this particular embodiment, the assays described above
are performed over time and the variation in the level of PCA3
nucleic acid and of another prostate specific marker (e.g., PSA
mRNA) present in the sample (e.g., urine sample) is evaluated. In
general, prostate cancer is considered as progressing when the
relative (i.e. relatively to the amount of cells or cell components
(e.g., protein or nucleic acids present therein) level of PCA3
nucleic acid detected increases with time. In contrast a cancer is
not considered as progressing when the relative level of PCA3
nucleic acid either decreases or remains constant over time.
[0091] One skilled in the art can select the nucleic acid primers
according to techniques known in the art as described above.
Samples to be tested include but should not be limited to RNA
samples from human tissue.
[0092] In a related aspect, it is possible to verify the efficiency
of nucleic acid amplification and/or detection only, by performing
external control reaction(s) using highly purified control target
nucleic acids added to the amplification and/or detection reaction
mixture. Alternatively, the efficiency of nucleic acid recovery
from cells and/or organelles, the level of nucleic acid
amplification and/or detection inhibition (if present) can be
verified and estimated by adding to each test sample control cells
or organelles (e.g., a define number of cells from a prostate
cancer cell line expressing PCA3 and second marker) by comparison
with external control reaction(s). To verify the efficiency of
both, sample preparation and amplification and/or detection, such
external control reaction(s) may be performed using a reference
test sample or a blank sample spiked with cells, organelles and/or
viral particles carrying the control nucleic acid sequence(s). For
example, a signal from the internal control (IC) sequences present
into the cells, viruses and/or organelles added to each test sample
that is lower than the signal observed with the external control
reaction(s) may be explained by incomplete lysis and/or inhibition
of the amplification and/or detection processes for a given test
sample. On the other hand, a signal from the IC sequences that is
similar to the signal observed with the external control
reaction(s), would confirm that the sample preparation including
cell lysis is efficient and that there is no significant inhibition
of the amplification and/or detection processes for a given test
sample. Alternatively, verification of the efficiency of sample
preparation only may be performed using external control(s)
analyzed by methods other than nucleic acid testing (e.g. analysis
using microscopy, mass spectrometry or immunological assays).
[0093] Therefore, in one particular embodiment, the methods of the
present invention uses purified nucleic acids, prostate cells or
viral particles containing nucleic acid sequences serving as
targets for an internal control (IC) in nucleic acid test assays to
verify the efficiency of cell lysis and of sample preparation as
well as the performance of nucleic acid amplification and/or
detection. More broadly, the IC serves to verify any chosen step of
the process of the present invention.
[0094] IC in PCR or related amplification techniques can be highly
purified plasmid DNA either supercoiled, or linearized by digestion
with a restriction endonuclease and repurified. Supercoiled IC
templates are amplified much less efficiently (about 100 fold) and
in a less reproducible manner than linearized and repurified IC
nucleic acid templates. Consequently, IC controls for amplification
and detection of the present invention are preferably performed
with linearized and repurified IC nucleic acid templates when such
types of IC are used.
[0095] The nucleic acids, cells, and/or organelles are incorporated
into each test sample at the appropriate concentration to obtain an
efficient and reproducible amplification/detection of the IC, based
on testing during the assay optimization. The optimal number of
control cells added, which is dependent on the assay, is
preferentially the minimal number of cells which allows a highly
reproducible IC detection signal without having any significant
detrimental effect on the amplification and/or detection of the
other genetic target(s) of the nucleic acid-based assay. A sample
to which is added the purified linearized nucleic acids, cells,
viral particles or organelles is generally referred to as a "spiked
sample".
[0096] Within certain embodiments, the amount of mRNA may be
detected via a RT-PCR based assay. In RT-PCR, the polymerase chain
reaction (PCR) is applied in conjunction with reverse
transcription. In such an assay, at least two oligonucleotide
primers may be used to amplify a portion of PCA3 cDNA derived from
a biological sample, wherein at least one oligonucleotide is
specific for (i.e. hybridizes to) a PCA3 RNA. The amplified cDNA
may then be separated and detected using techniques that are well
known in the art such as gel electrophoresis and ethidium bromide
staining. Amplification may be performed on biological samples
taken from a test patient and an individual who is not afflicted
with a prostate cancer (control sample), or using other types of
control samples. The amplification reaction may be performed on
several dilutions of cDNA (or directly on several dilutions of the
biological sample) spanning, for example, two orders of magnitude.
A value above a predetermined cut off value is indicative of the
presence or predisposition to develop prostate cancer. In general,
the elevated expression of PCA3 nucleic acid in a biological sample
as compared to control samples indicates the presence or the
predisposition to develop prostate cancer.
[0097] In further embodiments, PCA3 RNA is detected in a nucleic
acid extract from a biological sample by an in vitro RNA
amplification method named Nucleic Acid Sequence-Based
Amplification (NASBA). Other mRNA amplification methods well known
in the art may also be used and include transcriptase-mediated
amplification (TMA), strand displacement amplification (SDA), the
Q.beta. replicase system and Ligase chain reaction (LCR) (see
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25 and (Kwoh
et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177; Lizardi et
al., 1988, BioTechnology 6:1197-1202; Malek et al., 1994, Methods
Mol. Biol., 28:253-260; and Sambrook et al., 2000, Molecular
Cloning--A Laboratory Manual, Third Edition, CSH Laboratories).
[0098] The amplification and/or detection of prostate cancer
specific PCA3 RNA sequences and of the prostate specific marker can
be carried out simultaneously (e.g., multiplex real-time
amplification assays.)
[0099] Alternatively, oligonucleotide probes that specifically
hybridize under stringent conditions to a PCA3 nucleic acid may be
used in a nucleic acid hybridization assay (e.g., Southern and
Northern blots, dot blot, slot blot, in situ hybridization and the
like) to determine the presence and/or amount of prostate cancer
specific PCA3 polynucleotide in a biological sample.
[0100] Alternatively, oligonucleotides and primers could be
designed to directly sequence and assess the presence of prostate
cancer specific PCA3 sequences in the patient sample following an
amplification step. Such sequencing-based diagnostic methods are
automatable and are encompassed by the present invention.
[0101] 1. Synthesis of Nucleic Acid
[0102] The nucleic acid (e.g. DNA or RNA) for practicing the
present invention may be obtained according to well known
methods.
[0103] Isolated nucleic acid molecules of the present invention are
meant to include those obtained by cloning as well as those
chemically synthesized. Similarly, an oligomer which corresponds to
the nucleic acid molecule, or to each of the divided fragments, can
be synthesized. Such synthetic oligonucleotides can be prepared,
for example, by the triester method of Matteucci et al., J. Am.
Chem. Soc. 103:3185-3191 (1981) or by using an automated DNA
synthesizer.
[0104] An oligonucleotide can be derived synthetically or by
cloning. If necessary, the 5'-ends of the oligomers can be
phosphorylated using T4 polynucleotide kinase. Kinasing of single
strands prior to annealing or for labeling can be achieved using an
excess of the enzyme. If kinasing is for the labeling of probe, the
ATP can contain high specific activity radioisotopes. Then, the DNA
oligomer can be subjected to annealing and ligation with T4 ligase
or the like. Of course the labeling of a nucleic acid sequence can
be carried out by other methods known in the art.
[0105] II. Probes and Primers
[0106] The present invention relates to a nucleic acid for the
specific detection, in a sample, of the presence of PCA3 nucleic
acid sequences which are associated with prostate cancer,
comprising the above-described nucleic acid molecules or at least a
fragment thereof which binds under stringent conditions to PCA3
nucleic acid.
[0107] In one preferred embodiment, the present invention relates
to oligos which specifically target and enable amplification (i.e.
primers) of PCA3 RNA sequences associated with prostate cancer.
[0108] In another embodiment, PCA3 RNA can be detected using a
specific probe in an hybridization assay (e.g. Northern blot, dot
blot, slot blot and the like).
[0109] Oligonucleotide probes or primers of the present invention
may be of any suitable length, depending on the particular assay
format and the particular needs and targeted sequences employed. In
a preferred embodiment, the oligonucleotide probes or primers are
at least 10 nucleotides in length (preferably, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32 . . . ) and they may be adapted to be especially suited for a
chosen nucleic acid amplification system. Longer probes and primers
are also within the scope of the present invention as well known in
the art. Primers having more than 30, more than 40, more than 50
nucleotides and probes having more than 100, more than 200, more
than 300, more than 500 more than 800 and more than 1000
nucleotides in length are also covered by the present invention. Of
course, longer primers have the disadvantage of being more
expensive and thus, primers having between 12 and 30 nucleotides in
length are usually designed and used in the art. As well known in
the art, probes ranging from 10 to more than 2000 nucleotides in
length can be used in the methods of the present invention. As for
the % of identity described above, non-specifically described sizes
of probes and primers (e.g., 16, 17, 31, 24, 39, 350, 450, 550,
900, 1240 nucleotides, . . . ) are also within the scope of the
present invention. In one embodiment, the oligonucleotide probes or
primers of the present invention specifically hybridize with a PCA3
RNA (or its complementary sequence). More preferably, the primers
and probes will be chosen to detect a PCA3 RNA which is associated
with prostate cancer. In one embodiment, the probes and primers
used in the present invention do not hybridize with the PCA3 gene
(i.e. enable the distinction gene and expressed PCA3). Other
primers of the present invention are specific for a second
prostate-specific marker such as PSA (SEQ ID NO 11). Of course
othervariants well known in the art can also be used (U.S. Pat.
Nos. 6,479,263 and 5,674,682) as second prostate specific marker.
Because of the structural and sequence similarities of the PSA gene
with other members of the kallikrein gene family, the appropriate
selection of PSA sequences to serve as PSA-specific probes or
primers is critical to methods of amplification and/or detection of
PSA specific nucleic acids. Examples of suitable primers for PSA,
hK2/KLK2, PSMA, amplification and detection (e.g., U.S. Pat. No.
6,551,778) are well known in the art as well as for
transglutaminase 4, acid phosphatase and PCGEM1. In one embodiment,
the PSA oligonucleotide may also hybridize to other kallikrein
family members such as kallikrein 2 (hK2/hKLK2). One example of
such oligonucleotide is SEQ ID no 12.
[0110] As commonly known in the art, the oligonucleotide probes and
primers can be designed by taking into consideration the melting
point of hybridization thereof with its targeted sequence (see
below and in Sambrook et al., 1989, Molecular Cloning--A Laboratory
Manual, 2nd Edition, CSH Laboratories; Ausubel et al., 1994, in
Current Protocols in Molecular Biology, John Wiley & Sons Inc.,
N.Y.).
[0111] To enable hybridization to occur under the assay conditions
of the present invention, oligonucleotide primers and probes should
comprise an oligonucleotide sequence that has at least 70% (at
least 71%, 72%, 73%, 74%), preferably at least 75% (75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%) and
more preferably at least 90% (90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 100%) identity to a portion of a PCA3
polynucleotide. Probes and primers of the present invention are
those that hybridize to PCA3 nucleic acid (e.g. cDNA or mRNA)
sequence under stringent hybridization conditions and those that
hybridize to PCA3 gene homologs under at least moderately stringent
conditions. In certain embodiments probes and primers of the
present invention have complete sequence identity to PCA3 gene
sequence (e.g. cDNA or mRNA). However, probes and primers differing
from the native PCA3 gene sequence that keep the ability to
hybridize to native PCA3 gene sequence under stringent conditions
may also be used in the present invention. It should be understood
that other probes and primers could be easily designed and used in
the present invention based on the PCA3 nucleic acid sequence
disclosed herein (SEQ ID NOs 9, 10 and 13) by using methods of
computer alignment and sequence analysis known in the art (cf.
Molecular Cloning: A Laboratory Manual, Third Edition, edited by
Cold Spring Harbor Laboratory, 2000).
[0112] For example, a primer can be designed so as to be
complementary to a short PCA3 RNA which is associated with a
malignant state of the prostate cancer, whereas a long PCA3 RNA is
associated with a non-malignant state (benign) thereof
(PCT/CA00/01154 published under No. WO 01/23550). In accordance
with the present invention, the use of such a primer with the other
necessary reagents would give rise to an amplification product only
when a short PCA3 RNA (e.g., SEQ ID NO: 8) associated with prostate
cancer is present in the sample. The longer PCA3 (e.g., SEQ ID NO:
7) would not give rise to an amplicon. Of course, the amplification
could be designed so as to amplify a short and a long PCA3 mRNA. In
such a format, the long PCA3 mRNA could be used as the second
prostate specific marker.
[0113] In an embodiment as described above, the quantification of
the amplification products of short versus long PCA3 could be
carried out together with the detection of another prostate
specific marker to serve as a molecular diagnostic test for
prostate cancer. In another embodiment, primer pairs (or probes)
specific for PCA3 could be designed to avoid the detection of the
PCA3 gene or of unspliced PCA3 RNA. For example, the primers
sequences to be used in the present invention could span two
contiguous exons so that it cannot hybridize to an exon/intron
junction of the PCA3 gene. The amplification product obtained by
the use of such primer would be intron less between two chosen
exons (for examples of such primers and probes see table 1 and 2
below). Therefore, unspliced variants and genomic DNA would not be
amplified. It will be recognized by the person of ordinary skill
that numerous probes can be designed and used in accordance with a
number of embodiments of the present invention. Such tests can be
adapted using the sequence of PCA3 and that of the second
prostate-specific marker. Of course, different primer pairs (and
probes) can be designed from any part of the PCA3 sequences (SEQ ID
NOs: 7, 8, 9, 10 and 13) as well as from the sequence of PSA
(genbank accession number M27274, SEQ ID NO 11) or any other chosen
second prostate specific marker (e.g.,KLK2 (genbank acc. No.
NM005551), PSMA (genbank acc. No.BC025672), transglutaminase 4
(genbank acc. No.BC007003), acid phosphatase (genbank acc. No.
BC016344), PCGEM 1 (genbank acc. No. AF223389)).
[0114] Probes of the invention can be utilized with naturally
occurring sugar-phosphate backbones as well as modified backbones
including phosphorothioates, dithionates, alkyl phosphonates and
.alpha.-nucleotides and the like. Modified sugar-phosphate
backbones are generally taught by Miller, 1988, Ann. Reports Med.
Chem. 23:295 and Moran et al., 1987, Nucleic Acids Res., 14:5019.
Probes of the invention can be constructed of either ribonucleic
acid (RNA) or deoxyribonucleic acid (DNA), and preferably of
DNA.
[0115] Although the present invention is not specifically dependent
on the use of a label for the detection of a particular nucleic
acid sequence, such a label might be beneficial, by increasing the
sensitivity of the detection. Furthermore, it enables automation.
Probes can be labeled according to numerous well-known methods
(Sambrook et al., 2000, supra). Non-limiting examples of detectable
markers and labels include .sup.3H, .sup.14C, .sup.32P, and
.sup.35S, ligands, fluorophores, chemiluminescent agents, enzymes,
and antibodies. Other detectable markers for use with probes, which
can enable an increase in sensitivity of the method of the
invention, include biotin and radionucleotides. It will become
evident to the person of ordinary skill that the choice of a
particular label dictates the manner in which it is bound to the
probe.
[0116] As commonly known, radioactive nucleotides can be
incorporated into probes of the invention by several methods.
Non-limiting examples thereof include kinasing the 5' ends of the
probes using gamma .sup.32P ATP and polynucleotide kinase, using
the Klenow fragment of Pol I of E. coli in the presence of
radioactive dNTP (e.g. uniformly labeled DNA probe using random
oligonucleotide primers), using the SP6/T7 system to transcribe a
DNA segment in the presence of one or more radioactive NTP, and the
like.
[0117] In one embodiment, the label used in a homogenous detection
assay is a chemiluminescent compound (e.g., U.S. Pat. Nos.
5,656,207, 5,658,737 and 5,639,604), more preferably an acridinium
ester ("AE") compound, such as standard AE or derivatives thereof.
Methods of attaching labels to nucleic acids and detecting
labels-are well known (e.g., see Sambrook et al., Molecular
Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor
Laboratory Press, Cold Spring Habor, N.Y., 1989), Chapt. 10; U.S.
Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174 and 4,581,333;
and European Pat. App. No.0 747 706). Preferred methods of labeling
a probe with an AE compound attached via a linker have been
previously described detail (e.g., see U.S. Pat. No. 5,639,604,
Example 8).
[0118] Amplification of a selected, or target, nucleic acid
sequence may be carried out by a number of suitable methods. See
generally Kwoh et al., 1990, Am. Biotechnol. Lab. 8:14-25. Numerous
amplification techniques have been described and can be readily
adapted to suit particular needs of a person of ordinary skill.
Non-limiting examples of amplification techniques include
polymerase chain reaction (PCR, RT PCR . . . ), ligase chain
reaction (LCR), strand displacement amplification (SDA),
transcription-based amplification, the Q.beta. replicase system and
NASBA (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86, 1173-1177;
Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek et al.,
1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000,
supra). Other non-limiting examples of amplification methods
include rolling circle amplification (RCA); signal mediated
amplification of RNA technology (SMART); split complex
amplification reaction (SCAR); split promoter amplification of RNA
(SPAR).
[0119] Non-limiting examples of suitable methods to detect the
presence of the amplified products include the followings: agarose
or polyacrylamide gel, addition of DNA labeling dye in the
amplification reaction (such as ethidium bromide, picogreen, SYBER
green, etc.) and detection with suitable apparatus (fluorometer in
most cases). Other suitable methods include sequencing reaction
(either manual or automated); restriction analysis (provided
restriction sites were built into the amplified sequences), or any
method involving hybridization with a sequence specific probe
(Southern or Northern blot, TaqMan.TM. probes, molecular beacons,
and the like). Of course, other amplification methods are
encompassed by the present invention. Molecular beacons are
exemplified herein as one method for detecting the amplified
products according to the present invention (see below).
[0120] Of course in some embodiment direct detection (e.g.,
sequencing) of PCA3 cancer specific sequences as well as that of
another prostate specific marker in a sample may be performed using
specific probes or primers.
[0121] In one embodiment, the present invention has taken advantage
of technological advances in methods for detecting and identifying
nucleic acids. Therefore, the present invention is suitable for
detection by one of these tools called molecular beacons.
[0122] Molecular beacons are single-stranded oligonucleotide
hybridization probes/primers that form a stem loop structure. The
loop contains a probe sequence that is complementary to a target
sequence, and the stem is formed by the annealing of complementary
arm sequences that are located on either side of the probe/primer
sequence. A fluorophore is covalently linked to the end of one arm
and a quencher is covalently linked to the end of the other arm.
Molecular beacons do not fluoresce when they are free in solution.
However, when they hybridize to a nucleic acid strand containing a
target sequence they undergo comformational change that enables
them to fluoresce brightly (see U.S. Pat. Nos. 5,925,517, and
6,037,130). Molecular beacons can be used as amplicon detector
probes/primers in diagnostic assays. Because nonhybridized
molecular beacons are dark, it is not necessary to isolate the
probe-target hybrids to determine for example, the number of
amplicons synthesized during an assay. Therefore, molecular beacons
simplify the manipulations that are often required when traditional
detection and identifications means are used.
[0123] By using different colored fluorophores, molecular beacons
can also be used in multiplex amplification assays such as assays
that target the simultaneous amplification and detection of PCA3
nucleic acid and of the second specific prostate nucleic acid
(e.g., PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase
and PCGEM1). The design of molecular beacons probes/primers is well
known in the art and softwares dedicated to help their design are
commercially available (e.g., Beacon designer from Premier Biosoft
International). Molecular beacon probes/primers can be used in a
variety of hybridization and amplification assays (e.g., NASBA and
PCR).
[0124] In accordance with one embodiment of the present invention,
the amplified product can either be directly detected using
molecular beacons as primers for the amplification assay (e.g.,
real-time multiplex NASBA or PCR assays) or indirectly using,
internal to the primer pair binding sites, a molecular beacon probe
of 18 to 25 nucleotides long (e.g., 18, 19, 20, 21, 22, 23, 24, 25)
wich specifically hybridizes to the amplification product.
Molecular beacons probes or primers having a length comprised
between 18 and 25 nucleotides are preferred when used according to
the present invention (Tyagi et al., 1996, Nature Biotechnol. 14:
303-308). Shorter fragments could result in a less fluorescent
signal, whereas longer fragments often do not increase
significantly the signal. Of course shorter or longer probes and
primers could nevertheless be used.
[0125] Examples of nucleic acid primers which can be derived from
PCA3 RNA sequences are shown hereinbelow in Table 1:
1TABLE 1 NUCLEIC ACID PRIMERS Size (no. of bases) Nucleotides Exon
1 98 1-98 of SEQ ID NO: 9 Exon 2 165 99-263 of SEQ ID NO: 9 Exon 3
183 264-446 of SEQ ID NO: 9 Exon 4a 539 447-985 of SEQ ID NO: 9
Exon 4b 1052 986-2037 of SEQ ID NO: 9 Exon 1 120 1-120 of SEQ ID
NO: 10 Exon 2 165 121-285 of SEQ ID NO: 10 Exon 3 183 286-468 of
SEQ ID NO: 10 Exon 4a 539 469-1007 of SEQ ID NO: 10 Exon 4b 1059
1008-2066 of SEQ ID NO: 10 Exon 4c 556 2067-2622 of SEQ ID NO: 10
Exon 4d 960 2623-3582 of SEQ ID NO: 10 Exon junction 1 20 89-108 of
SEQ ID NO: 9 Exon junction 1 20 109-128 of SEQ ID NO: 10 Exon
junction 2 20 252-271 of SEQ ID NO: 9 Exon junction 2 20 274-293 of
SEQ ID NO: 10 Exon junction 3 20 435-454 of SEQ ID NO: 9 Exon
junction 3 20 457-476 of SEQ ID NO: 10 Exon junction 4 20 974-993
of SEQ ID NO: 9 Exon junction 4 20 996-1015 of SEQ ID NO: 10 Exon
junction 5 20 2055-2074 of SEQ ID NO: 10 Exon junction 6 20
2611-2630 of SEQ ID NO: 10
[0126] It should be understood that the sequences and sizes of the
primers taught in Table 1 are arbitrary and that a multitude of
other sequences can be designed and used in accordance with the
present invention.
[0127] While the present invention can be carried out without the
use of a probe which targets PCA3 sequences, such as the exon
junctions of PCA3 in accordance with the present invention, such
probes can add a further specificity to the methods and kits of the
present invention. Examples of specific nucleic acid probes which
can be used in the present invention (and designed based on the
exonic sequences shown in Table 1) are set forth in Table 2,
below:
2TABLE 2 NUCLEIC ACID PROBES Size (no. of bases) Nucleotides Probe
1 20 1-20 of SEQ ID NO: 9 Probe 2 30 1-30 of SEQ ID NO: 9 Probe 3
40 1-40 of SEQ ID NO: 9 Probe 4 20 1-20 of SEQ ID NO: 10 Probe 5 30
1-30 of SEQ ID NO: 10 Probe 6 40 1-40 of SEQ ID NO: 10 Probe 7 20
89-108 of SEQ ID NO: 9 Probe 8 30 114-143 of SEQ ID NO: 10 Probe 9
30 257-286 of SEQ ID NO: 9 Probe 10 20 284-303 of SEQ ID NO: 10
Probe 11 20 274-293 of SEQ ID NO: 9
[0128] Of course, as will be understood by the person of ordinary
skill, a multitude of additional probes can be designed from the
same or other region of SEQ ID NO. 9 as well as from SEQ ID NO. 10
and 13 and other sequences of the present invention, whether they
target exon junctions or not. It will be clear that the sizes of
the probes taught in Table 2 are arbitrary and that a multitude of
other sequences can be designed and used in accordance with the
present invention.
[0129] It will be readily recognized by the person of ordinary
skill, that the nucleic acid sequences of the present invention
(e.g., probes and primers) can be incorporated into anyone of
numerous established kit formats which are well known in the
art.
[0130] In one embodiment of the above-described method, a nucleic
acid probe is immobilized on a solid support. Examples of such
solid supports include, but are not limited to, plastics such as
polycarbonate, complex carbohydrates such as agarose and sepharose,
and acrylic resins, such as polyacrylamide and latex beads.
Techniques for coupling nucleic acid probes to such solid supports
are well known in the art.
[0131] The test samples suitable for nucleic acid probing methods
of the present invention include, for example, cells or nucleic
acid extracts of cells, or biological fluids (e.g., urine). The
sample used in the above-described methods will vary based on the
assay format, the detection method and the nature of the tissues,
cells or extracts to be assayed. Methods for preparing nucleic acid
extracts of cells are well known in the art and can be readily
adapted in order to obtain a sample which is compatible with the
method utilized. Preferably the sample is a urine sample. When the
urine sample is used, it should contain at least one prostate cell
in order to enable the identification of the prostate specific
marker of the present invention. In fact, assuming that the
half-life of PCA3 mRNA in an untreated biological sample is not
suitable for easily enabling the preservation of the integrity of
its sequence, the collected sample, whether urine or otherwise,
should, prior to a treatment thereof contain at least one prostate
cell. It will be recognized that the number of cells in the sample
will have an impact on the validation of the test and on the
relative level of measured PCA3 (or second prostate specific
marker).
[0132] III. A Kit for Detecting the Presence of PCA3 Nucleic Acid
in a Sample
[0133] In another embodiment, the present invention relates to a
kit for diagnosing prostate cancer in a manner which is both
sensitive and specific (i.e lowering the number of false
positives). Such kit generally comprises a first container means
having disposed therein at least one oligonucleotide probe or
primer that hybridizes to a prostate cancer-specific PCA3 nucleic
acid sequence. In one embodiment, the present invention also
relates to a kit further comprising in a second container means
oligonucleotide probes or primers which are specific to a second
prostate specific marker, thereby validating a negative result with
PCA3.
[0134] In a particular embodiment of the present invention, this
kit (K) comprises a primer pair which enables the amplification of
PSA, hK2/KLK2, PSMA, transglutaminase 4, acid phosphatase and
PCGEM1) Of course the present invention also encompasses the use of
a third prostate specific marker.
[0135] Oligonucleotides (probes or primers) of the kit may be used,
for example, within a NASBA, PCR or hybridization assay.
Amplification assays may be adapted for real time detection of
multiple amplification products (i.e. multiplex real time
amplification assays).
[0136] In a related particular embodiment, the kit further includes
other containers comprising additional components such as
additional oligonucleotide or primer and/or one or more of the
following: buffers, reagents to be used in the assay (e.g. wash
reagents, polymerases or else) and reagents capable of detecting
the presence of bound nucleic acid probe or primers. Examples of
detection reagents include, but are not limited to radiolabelled
probes, enzymatic labeled probes (horse radish peroxidase, alkaline
phosphatase), and affinity labeled probes (biotin, avidin, or
steptavidin). In one embodiment, the detection reagents are
molecular beacon probes which specifically hybridizes to the
amplification products. In another embodiment, the detection
reagents are chemiluminescent compounds such as Acridinium Ester
(AE).
[0137] For example, a compartmentalized kit in accordance with the
present invention includes any kit in which reagents are contained
in separate containers. Such containers include small glass
containers, plastic containers or strips of plastic or paper. Such
containers allow the efficient transfer of reagents from one
compartment to another compartment such that the samples and
reagents are not cross contaminated and the agents or solutions of
each container can be added in a quantitative fashion from one
compartment to another. Such containers will include a container
which will accept the test sample (e.g., an RNA extract from a
biological sample or cells), a container which contains the primers
used in the assay, containers which contain enzymes, containers
which contain wash reagents, and containers which contain the
reagents used to detect the extension products. As mentioned above,
the separation or combination of reagents can be adapted by the
person of ordinary skill to which this invention pertain, according
to the type of kit which is preferred (e.g., a diagnostic kit based
on amplification or hybridization methods or both), the types of
reagents used and their stability or other intrinsic properties. In
one embodiment, one container contains the amplification reagents
and a separate container contains the detection reagent. In another
embodiment, amplification and detection reagents are contained in
the same container.
[0138] Kits may also contain oligonucleotides that serve as capture
oligomers for purifying the target nucleic acids from a sample.
Examples of capture oligomers have sequences of at least 15
nucleotides complementary to a portion of the PCA3 target nucleic
acid. Embodiments of capture oligomers may have additional bases
attached to a 3' or 5' end the sequence that is complementary to
the PCA3 target sequence which may act functionally in a
hybridization step for capturing the target nucleic acid. Such
additional sequences are preferably a homopolymeric tail sequence,
such as a poly-A or poly-T sequence, although other embodiments of
tail sequences are included in capture oligomers of the present
invention. In one embodiment, CAP binding protein (e.g., eIF4G-4E)
or part thereof may be used to capture cap-structure containing
mRNAs (Edery et al., 1987, Gene 74(2): 517-525). In another
embodiment, a non specific capture reagent is used (e.g., silica
beads).
[0139] Kits useful for practicing the methods of the present
invention may include those that include any of the amplification
oligonucleotides and/or detection probes disclosed herein which are
packaged in combination with each other. Kits may also include
capture oligomers for purifying the PCA3 target nucleic acid from a
sample, which capture oligomers may be packaged in combination with
the amplification oligonucleotides and/or detection probes.
[0140] In a further embodiment, cells contained in voided urine
samples obtained after an attentive digital rectal examination are
harvested and lysed in a lysis buffer. Nucleic acids are extracted
(e.g., from the lysate by solid phase extraction on silica beads
for example). Detection of the presence of RNA encoded by the PCA3
gene in the nucleic acid extract is done by an in vitro specific
RNA amplification coupled to real-time detection of amplified
products byfluorescent specific probes. In this method,
simultaneously to the amplification of the PCA3 prostate cancer
specific RNA undergoes the amplification of the second
prostate-specific marker (such as the PSA RNA) as a control for the
presence in the urine sample of prostate cells.
[0141] The screening and diagnostic methods of the invention do not
require that the entire PCA3 RNA sequence be detected. Rather, it
is only necessary to detect a fragment or length of nucleic acid
that is sufficient to detect the presence of the PCA3 nucleic acid
from a normal or affected individual, the absence of such nucleic
acid, or an altered structure of such nucleic acid (such as an
aberrant splicing pattern). For this purpose, any of the probes or
primers as described above are used, and many more can be designed
as conventionally known in the art based on the sequences described
herein and others known in the art.
[0142] It is to be understood that although the following
discussion is specifically directed to human patients, the
teachings are also applicable to any animal that expresses
PCA3.
[0143] The diagnostic and screening methods of the invention are
especially useful for a patient suspected of being at risk for
developing a disease associated with an altered expression level of
PCA3 based on family history, or a patient in which it is desired
to diagnose a PCA3-related disease (ex. prostate cancer). The
method of the present invention may also be used to monitor the
progression of prostate cancer in patient as described above.
[0144] The present invention is illustrated in further details by
the following non-limiting example. The examples are provided for
illustration only and should not be construed as limiting the scope
of the invention.
EXAMPLE 1
CLINICAL PERFORMANCE USING ONE ILLUSTRATIVE EMBODIMENT OF THE
METHODS OF THE PRESENT INVENTION
[0145] To estimate the clinical performance of the method, a pilot
study was done on 517 patients planned to undergo ultrasound guided
needle biopsies coming from five university medical centers located
in Montreal and Quebec (Canada) between September 2001 and June
2002. Each sample was processed using the following steps:
[0146] Sample Collection
[0147] Following an attentive digital rectal examination, the first
20 to 30 ml of voided urine was collected in sterile 80 ml plastic
containers (patient urinates directly in the sterile
container).
[0148] An equal volume of Sample buffer (0.1M phosphate (0.06M
Na.sub.2HPO.sub.4, 0.04M NaH.sub.2PO.sub.4) 0.3M NaCl, pH 7.0,) was
immediately added and the solution mixed by inversion.
[0149] If not processed immediately, samples were refrigerated
between 2-8.degree. C. for up to three days until further
processing. In view of the cell recovery step, freezing should be
avoided.
[0150] Cell Recovery
[0151] The sample was mixed by inversion; and the container gently
tapped on the counter in order to detach cells from the inner walls
thereof. The sample was then transferred into one or two (if
necessary) conical polypropylene tubes (40 ml/tube).
[0152] The cells were pelleted by centrifugation in a tabletop
centrifuge at 1400 g for 15 minutes. Finally, the supernatant was
decanted and the cells were immediately lysed.
[0153] Cell Lysis
[0154] 400 .mu.l of Lysis Buffer (4.68M GUSCN, 20 mM EDTA, 1.2%
Triton X-100.TM., 46 mM Tris-HCl, pH 7.2) was added to the urine
cell pellet.
[0155] The cell pellet was then vigorously vortexed for 20 seconds
in order to lyse the cells. It is important to make sure that no
particulate matter is left. The lysate was transparent and not too
viscous.
[0156] The lysate was transferred into a 1.5 ml microtubes and
vortexed for 30 seconds.
[0157] If desired, the lysed cells can now be stored at
.ltoreq.-70.degree. C. indefinitely.
[0158] Nucleic Acid Extraction
[0159] The silica suspension (60 g silica type .+-.80% particle
size 1-5 .mu.m, add MilliQ water at a final volume of 500 ml) was
first vigorously vortexed for 30 seconds until an opaque
homogeneous suspension was obtained.
[0160] 200 .mu.l of the suspension was then immediately removed and
added to the lysed specimen. All tubes were subsequently vigorously
vortexed for 15 seconds to bind nucleic acids to the silica.
[0161] On a test tube rack, a series of Microspin.TM. Columns
identifying each filter unit with the appropriate number of patient
were prepared.
[0162] The content of each microtube containing the lysed cells and
the silica were transferred into the membrane filter unit of one
Microspin.TM. Column. To facilitate the transfer of the particulate
matter, the microtube was vortexed briefly (approximately 5
seconds) in order to resuspend the content. The same was done
before transferring. Tips were changed between samples.
[0163] The Microspin.TM. columns were centrifuged in a
non-refrigerated microcentrifuge at 10,000 RPM for 5 minutes at
room temperature (18.degree. C.-25.degree. C.). The membrane filter
retained silica-bound nucleic acids whereas other cellular
components remained in the flow-through.
[0164] Meanwhile, a series of 2 ml microtubes corresponding to the
number of Microspin.TM. columns were prepared.
[0165] The membrane filter units containing the silica were
transferred to new 2 mL microtubes. 500 .mu.l of Wash Buffer (5.3M
GuSCN, 52 mM Tris-HCl pH 6.4) was added to each membrane filter
unit. The Microspin.TM. columns were then centrifuged in a
non-refrigerated microfuge at 10,000 RPM for 5 minutes at room
temperature.
[0166] On a test tube rack, a new series of 2 ml microtubes were
prepared.
[0167] The membrane filter units with the silica were transferred
to the new 2 ml microtubes. 600 .mu.l ethanol 70% was added to the
membrane filter units. The Microspin.TM. columns were then
centrifuged in a non-refrigerated microfuge at 10,000 RPM for 5
minutes at room temperature.
[0168] On a test tube rack, a new series of 2 ml microtubes were
prepared.
[0169] The membrane filter units with the silica are transferred to
a new 2 ml microtube. Discard the microtubes containing the
flow-through.
[0170] The membrane filter unit containing microtubes were then
transferred to a heating block at 65.degree. C..+-.1.degree. C.
installed under a fume hood.
[0171] All tubes were opened carefully to ensure evaporation, and
incubated for approximately 10 minutes to dry the silica.
[0172] 200 .mu.l Elution Buffer (Dnase/Rnase-free water) to each
membrane filter unit was added.
[0173] The membrane filter units were then centrifuged in a
microfuge at 10,000 RPM for 5 minutes at room temperature.
[0174] The elution steps were repeated once to obtain a second
eluate. These steps elute nucleic acids from the silica and
concentrate them in the flow-through.
[0175] The microfilter units were disposed and the two microtubes
containing the nucleic acid elution were kept.
[0176] For each eluate, three aliquots of .congruent.50 .mu.l of
nucleic acids were stored at .ltoreq.-70.degree. C.
[0177] In Vitro RNA Amplification and Detection
[0178] The nucleic acid eluate sample to test was first thawed on
ice. The reaction mix was then prepared according to the number of
reactions to be performed. Each sample was made at least in
duplicate.
[0179] 10 .mu.l of the reaction mix was distributed in identified
microtubes [80 mM Tris-HCl pH 8.5, 24 mM MgCl.sub.2, 180 mM KCl, 10
mM DTT, 2 mM of each dNTP, 4 mM of rATP, rUTP, CTP, 3 mM rGTP, 1 mM
ITP, 30% DMSO, 3% sucrose, 1% D-Mannitol, 1% Dextran T-40, 208 nM
PSA primers (N2psaP1B, SEQ. ID NO 1 and N2psaP2B, SEQ. ID NO 2),
417 nM PCA-3 primers (N0pcaP1A, SEQ. ID NO 3 and N0pcaP2B, SEQ. ID
NO 4), 84 nM PSA beacon (BpsaRD-4, SEQ. ID NO 5), 166 nM PCA-3
beacon (BpcaFD-4, SEQ. ID NO 6).
[0180] 5 .mu.l of nucleic acid sample eluate was added in each tube
and mixed.
[0181] Tubes were placed in a thermocycler.TM., heated at
65.degree. C..+-.1.degree. C. for a period of 5 minutes and then
the temperature was kept at 41.degree. C. After 5 minutes at
41.degree. C., tubes were retrieved and centrifuged briefly in
order to remove the condensation drops from the lids.
[0182] The next steps were better carried out quickly, and the tube
temperature was preferably kept at 41.degree. C.
[0183] 5 .mu.l of the enzyme mix (375 mM sorbitol, 0.105
.mu.g/.mu.l BSA, 0.08 units of RnaseH, 32.0 units of T7 RNA
polymerase, 6.4 units of AMV-RT) was then quickly added to each
tube and the tubes were gently mixed.
[0184] The tubes were put back into the EasyQ.TM. incubator. When
the last tube was in place, the incubator was kept at a temperature
of 41.degree. C.+-.0.5.degree. C. for 5 minutes.
[0185] The tubes were then briefly centrifuged. Quickly, all tubes
were transferred in a thermostated spectrofluorimeter for in vitro
RNA amplification and real time amplified product detection with
the following characteristics: (1) the light source was a
quartz-halogen lamp, (2) the filter used for ROX
(6-carboxy-x-rhodamine N-succinimidyl ester) fluorescence was at
550-620 nm and for FAM (6-carboxyfluorescein N-hydroxysuccinimide
ester) was at 485-530 nm, (3) the fluorescence integration time per
tube was 20 msec; and (4) ROX and FAM emission was read each 30 sec
and the tube block was set at the temperature of 41.degree.
C..+-.1.degree. C.
[0186] Results
[0187] Fluorescence data generated during the two hours of
amplification underwent fitting following the approach of Brown
[Computer Methods and Programs in Biomedicine 65 (2001)
191-200].
[0188] Based on the PSA ratio (fluo maxlfluo min) cut off of 1.3,
out of the 517 patients who have been tested, 443 had adequate
quantities of prostate cells in the urine.
[0189] In this population of patients, 34% (151/443) had prostate
cancer confirmed by histology.
3TABLE 3 Positive Biopsies versus tPSA Categories tPSA Percentage
of Patients Positive Biopsies <4 ng/ml 21% (n = 94) 20% (n = 19)
4-10 ng/ml 55% (n = 243) 35% (n = 85) >10 ng/ml 24% (n = 106)
44% (n = 47)
[0190] Clinical specificity (Sp) and sensitivity (Se) of the method
has been estimated following a tree-structured classification using
S-plus.TM. software [Insightful Corporation, Seattle, Wash., USA]
starting from raw fluorimeter data. Three structured trees have
been defined for the three types of patients defined as having a
total blood PSA (tPSA) below 4 ng/ml, between 4-10 ng/ml and above
10 ng/ml (see FIGS. 2-4 and TABLE 4.
4TABLE 4 Method sensitivity and specificity tPSA Number Se % Sp %
<4 ng/ml 94 74 (14/19) 91 (68/75) 4-10 ng/ml 243 59 (50/85) 91
(144/158) >10 ng/ml 106 79 (37/47) 80 (47/59) Overall 443 67
(101/151) 89 (259/292)
[0191]
5TABLE 5 Method performance versus total tPSA and free fPSA Se % Sp
% tPSA .gtoreq. 2.5 ng/ml 100% (58/58) 6% (5/88) tPSA .gtoreq. 4.0
ng/ml 88% (51/58) 15% (13/88) FPSA/tPSA .ltoreq. 0.15 72% (42/58)
56% (49/88) FPSA/tPSA .ltoreq. 0.13 66% (38/58) 67% (59/88) uPM3
.TM. 64% (37/58) 91% (80/88) 146/443 patients with available
fPSA
[0192] The study demonstrated that the method has a positive
predictive value (PPV) of 75%, compared to total PSA (>4.0
ng/ml) with a PPV of only 38%. The negative predictive value of the
method is 84%, compared to 81% for tPSA. The overall accuracy of
the method is 81%, compared with an accuracy of 47% for tPSA.
[0193] Although the present invention has been described
hereinabove by way of illustrative embodiments thereof, it will be
appreciated by one skilled in the art from reading of this
disclosure that various changes in form and detail can be made
without departing from the spirit and nature of the invention as
defined in the appended claims. For example, various other
amplification assays or detection assays, different probes and
primers sequences as well as slightly different temperature and
time of incubation may be used according to the present invention.
Sequence CWU 1
1
13 1 47 DNA Homo sapiens 1 aattctaata cgactcacta tagggaggat
gaaacaggct gtgccga 47 2 20 DNA Homo sapiens 2 agcattccca accctggcag
20 3 45 DNA Homo sapiens 3 aattctaata cgactcacta tagggcctgc
ccatccttta aggaa 45 4 20 DNA Homo sapiens 4 caggaagcac aaaaggaagc
20 5 24 DNA Homo sapiens 5 cccagtctgc ggcggtgttc tggg 24 6 28 DNA
Homo sapiens 6 cgcttgtgag ggaaggacat tagaagcg 28 7 506 DNA Homo
sapiens 7 caggaagcac aaaaggaagc acagaggtaa gtgctttata aagcactcaa
tttctactca 60 gaaatttttg atggccttaa gttcctctac tcgtttctat
ccttcctact cactgtcctc 120 ccggaatcca ctaccgattt tctatttctt
gcctcgtatt gtctgactgg ctcacttgga 180 tttatcctca cggagtctgg
attttctacc cgggctcacc tccgtccctc catatttgtc 240 ctccactttc
acagatccct gggagaaatg cccggccgcc atcttgggtc atcgatgagc 300
ctcgccctgt gcctggtccc gcttgtgagg gaaggacatt agaaaatgaa ttgatgtgtt
360 ccttaaagga tgggcaggaa aacagatcct gttgtggata tttatttgaa
cgggattaca 420 gatttgaaat gaagtcacca aagtgagcat taccaatgag
aggaaaacag acgagaaaat 480 cttgatggct tcacaagaca tgcaac 506 8 278
DNA Homo sapiens 8 caggaagcac aaaaggaagc acagagatcc ctgggagaaa
tgcccggccg ccatcttggg 60 tcatcgatga gcctcgccct gtgcctggtc
ccgcttgtga gggaaggaca ttagaaaatg 120 aattgatgtg ttccttaaag
gatgggcagg aaaacagatc ctgttgtgga tatttatttg 180 aacgggatta
cagatttgaa atgaagtcac caaagtgagc attaccaatg agaggaaaac 240
agacgagaaa atcttgatgg cttcacaaga catgcaac 278 9 2036 DNA Homo
sapiens misc_feature (1472)..(1472) n is a or c or g or t 9
agaagctggc atcagaaaaa cagaggggag atttgtgtgg ctgcagccga gggagaccag
60 gaagatctgc atggtgggaa ggacctgatg atacagagga attacaacac
atatacttag 120 tgtttcaatg aacaccaaga taaataagtg aagagctagt
ccgctgtgag tctcctcagt 180 gacacagggc tggatcacca tcgacggcac
tttctgagta ctcagtgcag caaagaaaga 240 ctacagacat ctcaatggca
ggggtgagaa ataagaaagg ctgctgactt taccatctga 300 ggccacacat
ctgctgaaat ggagataatt aacatcacta gaaacagcaa gatgacaata 360
taatgtctaa gtagtgacat gtttttgcac atttccagcc cctttaaata tccacacaca
420 caggaagcac aaaaggaagc acagagatcc ctgggagaaa tgcccggccg
ccatcttggg 480 tcatcgatga gcctcgccct gtgcctggtc ccgcttgtga
gggaaggaca ttagaaaatg 540 aattgatgtg ttccttaaag gatgggcagg
aaaacagatc ctgttgtgga tatttatttg 600 aacgggatta cagatttgaa
atgaagtcac aaagtgagca ttaccaatga gaggaaaaca 660 gacgagaaaa
tcttgatggc ttcacaagac atgcaacaaa caaaatggaa tactgtgatg 720
acatgaggca gccaagctgg ggaggagata accacggggc agagggtcag gattctggcc
780 ctgctgccta aactgtgcgt tcataaccaa atcatttcat atttctaacc
ctcaaaacaa 840 agctgttgta atatctgatc tctacggttc cttctgggcc
caacattctc catatatcca 900 gccacactca tttttaatat ttagttccca
gatctgtact gtgacctttc tacactgtag 960 aataacatta ctcattttgt
tcaaagaccc ttcgtgttgc tgcctaatat gtagctgact 1020 gtttttccta
aggagtgttc tggcccaggg gatctgtgaa caggctggga agcatctcaa 1080
gatctttcca gggttatact tactagcaca cagcatgatc attacggagt gaattatcta
1140 atcaacatca tcctcagtgt ctttgcccat actgaaattc atttcccact
tttgtgccca 1200 ttctcaagac ctcaaaatgt cattccatta atatcacagg
attaactttt ttttttaacc 1260 tggaagaatt caatgttaca tgcagctatg
ggaatttaat tacatatttt gttttccagt 1320 gcaaagatga ctaagtcctt
tatccctccc ctttgtttga ttttttttcc agtataaagt 1380 taaaatgctt
agccttgtac tgaggctgta tacagcacag cctctcccca tccctccagc 1440
cttatctgtc atcaccatca acccctccca tnysacctaa acaaaatcta acttgtaatt
1500 ccttgaacat gtcaggncat acattrttcc ttctgcctga gaagctcttc
cttgtctctt 1560 aantctagaa tgatgtaaag ttttgaataa gttgactatc
ttacttcatg caaagaaggg 1620 acacatatga gattcatcat ccatgagaca
gcaaatacta aaagtgtaat ttgattataa 1680 gagtttagat aaatatatga
aatgcaagak ccacagaggg aatgtttatg gggcacgttt 1740 gtaagcctgg
gatgtgaagm aaaggcaggg aacctcatag tatcttatat aatatacttc 1800
atttctctat ctctatcaca atatccaaca agcttttcac agaattcatg cagtgcaaat
1860 ccccaaaggt aacctttatc catttcatgg tgagtgcgct ttagaatttt
ggcaaatcat 1920 actggtcact tatctcaact ttgagatgtg tttgtccttg
tagttaattg aaagaaatag 1980 ggcactcttg tgagccactt tagggttcac
tcctggcaat aaagaattta caaaga 2036 10 3582 DNA Homo sapiens 10
acagaagaaa tagcaagtgc cgagaagctg gcatcagaaa aacagagggg agatttgtgt
60 ggctgcagcc gagggagacc aggaagatct gcatggtggg aaggacctga
tgatacagag 120 gaattacaac acatatactt agtgtttcaa tgaacaccaa
gataaataag tgaagagcta 180 gtccgctgtg agtctcctca gtgacacagg
gctggatcac catcgacggc actttctgag 240 tactcagtgc agcaaagaaa
gactacagac atctcaatgg caggggtgag aaataagaaa 300 ggctgctgac
tttaccatct gaggccacac atctgctgaa atggagataa ttaacatcac 360
tagaaacagc aagatgacaa tataatgtct aagtagtgac atgtttttgc acatttccag
420 cccctttaaa tatccacaca cacaggaagc acaaaaggaa gcacagagat
ccctgggaga 480 aatgcccggc cgccatcttg ggtcatcgat gagcctcgcc
ctgtgcctgg tcccgcttgt 540 gagggaagga cattagaaaa tgaattgatg
tgttccttaa aggatgggca ggaaaacaga 600 tcctgttgtg gatatttatt
tgaacgggat tacagatttg aaatgaagtc acaaagtgag 660 cattaccaat
gagaggaaaa cagacgagaa aatcttgatg gcttcacaag acatgcaaca 720
aacaaaatgg aatactgtga tgacatgagg cagccaagct ggggaggaga taaccacggg
780 gcagagggtc aggattctgg ccctgctgcc taaactgtgc gttcataacc
aaatcatttc 840 atatttctaa ccctcaaaac aaagctgttg taatatctga
tctctacggt tccttctggg 900 cccaacattc tccatatatc cagccacact
catttttaat atttagttcc cagatctgta 960 ctgtgacctt tctacactgt
agaataacat tactcatttt gttcaaagac ccttcgtgtt 1020 gctgcctaat
atgtagctga ctgtttttcc taaggagtgt tctggcccag gggatctgtg 1080
aacaggctgg gaagcatctc aagatctttc cagggttata cttactagca cacagcatga
1140 tcattacgga gtgaattatc taatcaacat catcctcagt gtctttgccc
atactgaaat 1200 tcatttccca cttttgtgcc cattctcaag acctcaaaat
gtcattccat taatatcaca 1260 ggattaactt ttttttttaa cctggaagaa
ttcaatgtta catgcagcta tgggaattta 1320 attacatatt ttgttttcca
gtgcaaagat gactaagtcc tttatccctc ccctttgttt 1380 gatttttttt
ccagtataaa gttaaaatgc ttagccttgt actgaggctg tatacagcac 1440
agcctctccc catccctcca gccttatctg tcatcaccat caacccctcc cataccacct
1500 aaacaaaatc taacttgtaa ttccttgaac atgtcaggac atacattatt
ccttctgcct 1560 gagaagctct tccttgtctc ttaaatctag aatgatgtaa
agttttgaat aagttgacta 1620 tcttacttca tgcaaagaag ggacacatat
gagattcatc atcacatgag acagcaaata 1680 ctaaaagtgt aatttgatta
taagagttta gataaatata tgaaatgcaa gagccacaga 1740 gggaatgttt
atggggcacg tttgtaagcc tgggatgtga agcaaaggca gggaacctca 1800
tagtatctta tataatatac ttcatttctc tatctctatc acaatatcca acaagctttt
1860 cacagaattc atgcagtgca aatccccaaa ggtaaccttt atccatttca
tggtgagtgc 1920 gctttagaat tttggcaaat catactggtc acttatctca
actttgagat gtgtttgtcc 1980 ttgtagttaa ttgaaagaaa tagggcactc
ttgtgagcca ctttagggtt cactcctggc 2040 aataaagaat ttacaaagag
ctactcagga ccagttgtta agagctctgt gtgtgtgtgt 2100 gtgtgtgtgt
gagtgtacat gccaaagtgt gcctctctct cttgacccat tatttcagac 2160
ttaaaacaag catgttttca aatggcacta tgagctgcca atgatgtatc accaccatat
2220 ctcattattc tccagtaaat gtgataataa tgtcatctgt taacataaaa
aaagtttgac 2280 ttcacaaaag cagctggaaa tggacaacca caatatgcat
aaatctaact cctaccatca 2340 gctacacact gcttgacata tattgttaga
agcacctcgc atttgtgggt tctcttaagc 2400 aaaatacttg cattaggtct
cagctggggc tgtgcatcag gcggtttgag aaatattcaa 2460 ttctcagcag
aagccagaat ttgaattccc tcatctttta ggaatcattt accaggtttg 2520
gagaggattc agacagctca ggtgctttca ctaatgtctc tgaacttctg tccctctttg
2580 tgttcatgga tagtccaata aataatgtta tctttgaact gatgctcata
ggagagaata 2640 taagaactct gagtgatatc aacattaggg attcaaagaa
atattagatt taagctcaca 2700 ctggtcaaaa ggaaccaaga tacaaagaac
tctgagctgt catcgtcccc atctctgtga 2760 gccacaacca acagcaggac
ccaacgcatg tctgagatcc ttaaatcaag gaaaccagtg 2820 tcatgagttg
aattctccta ttatggatgc tagcttctgg ccatctctgg ctctcctctt 2880
gacacatatt agcttctagc ctttgcttcc acgactttta tcttttctcc aacacatcgc
2940 ttaccaatcc tctctctgct ctgttgcttt ggacttcccc acaagaattt
caacgactct 3000 caagtctttt cttccatccc caccactaac ctgaattgcc
tagaccctta tttttattaa 3060 tttccaatag atgctgccta tgggctaata
ttgctttaga tgaacattag atatttaaag 3120 tctaagaggt tcaaaatcca
actcattatc ttctctttct ttcacctccc ctgctcctct 3180 ccctatatta
ctgattgact gaacaggatg gtccccaaga tgccagtcaa atgagaaacc 3240
cagtggctcc ttgtggatca tgcatgcaag actgctgaag ccagaggatg actgattacg
3300 cctcatgggt ggaggggacc actcctgggc cttcgtgatt gtcaggagca
agacctgaga 3360 tgctccctgc cttcagtgtc ctctgcatct cccctttcta
atgaagatcc atagaatttg 3420 ctacatttga gaattccaat taggaactca
catgttttat ctgccctatc aattttttaa 3480 acttgctgaa aattaagttt
tttcaaaatc tgtccttgta aattactttt tcttacagtg 3540 tcttggcata
ctatatcaac tttgattctt tgttacaact tt 3582 11 7130 DNA Homo sapiens
11 gaattccaca ttgtttgctg cacgttggat tttgaaatgc tagggaactt
tgggagactc 60 atatttctgg gctagaggat ctgtggacca caagatcttt
ttatgatgac agtagcaatg 120 tatctgtgga gctggattct gggttgggag
tgcaaggaaa agaatgtact aaatgccaag 180 acatctattt caggagcatg
aggaataaaa gttctagttt ctggtctcag agtggtgcag 240 ggatcaggga
gtctcacaat ctcctgagtg ctggtgtctt agggcacact gggtcttgga 300
gtgcaaagga tctaggcacg tgaggctttg tatgaagaat cggggatcgt acccaccccc
360 tgtttctgtt tcatcctggg catgtctcct ctgcctttgt cccctagatg
aagtctccat 420 gagctacaag ggcctggtgc atccagggtg atctagtaat
tgcagaacag caagtgctag 480 ctctccctcc ccttccacag ctctgggtgt
gggagggggt tgtccagcct ccagcagcat 540 ggggagggcc ttggtcagcc
tctgggtgcc agcagggcag gggcggagtc ctggggaatg 600 aaggttttat
agggctcctg ggggaggctc cccagcccca agcttaccac ctgcacccgg 660
agagctgtgt caccatgtgg gtcccggttg tcttcctcac cctgtccgtg acgtggattg
720 gtgagagggg ccatggttgg ggggatgcag gagagggagc cagccctgac
tgtcaagctg 780 aggctctttc ccccccaacc cagcacccca gcccagacag
ggagctgggc tcttttctgt 840 ctctcccagc cccacttcaa gcccataccc
ccagcccctc catattgcaa cagtcctcac 900 tcccacacca ggtccccgct
ccctcccact taccccagaa ctttctcccc attgcccagc 960 cagctccctg
ctcccagctg ctttactaaa ggggaagttc ctgggcatct ccgtgtttct 1020
ctttgtgggg ctcaaaacct ccaaggacct ctctcaatgc cattggttcc ttggaccgta
1080 tcactggtcc atctcctgag cccctcaatc ctatcacagt ctactgactt
ttcccattca 1140 gctgtgagtg tccaacccta tcccagagac cttgatgctt
ggcctcccaa tcttgcccta 1200 ggatacccag atgccaacca gacacctcct
tcttcctagc caggctatct ggcctgagac 1260 aacaaatggg tccctcagtc
tggcaatggg actctgagaa ctcctcattc cctgactctt 1320 agccccagac
tcttcattca gtggcccaca ttttccttag gaaaaacatg agcatcccca 1380
gccacaactg ccagctctct gattccccaa atctgcatcc ttttcaaaac ctaaaaacaa
1440 aaagaaaaac aaataaaaca aaaccaactc agaccagaac tgttttctca
acctgggact 1500 tcctaaactt tccaaaacct tcctcttcca gcaactgaac
ctggccataa ggcacttatc 1560 cctggttcct agcacccctt atcccctcag
aatccacaac ttgtaccaag tttcccttct 1620 cccagtccaa gaccccaaat
caccacaaag gacccaatcc ccagactcaa gatatggtct 1680 gggcgctgtc
ttgtgtctcc taccctgatc cctgggttca actctgctcc cagagcatga 1740
agcctctcca ccagcaccag ccaccaacct gcaaacctag ggaagattga cagaattccc
1800 agcctttccc agctccccct gcccatgtcc caggactccc agccttggtt
ctctgccccc 1860 gtgtcttttc aaacccacat cctaaatcca tctcctatcc
gagtccccca gttccccctg 1920 tcaaccctga ttcccctgat ctagcacccc
ctctgcaggc gctgcgcccc tcatcctgtc 1980 tcggattgtg ggaggctggg
agtgcgagaa gcattcccaa ccctggcagg tgcttgtggc 2040 ctctcgtggc
agggcagtct gcggcggtgt tctggtgcac ccccagtggg tcctcacagc 2100
tgcccactgc atcaggaagt gagtaggggc ctggggtctg gggagcaggt gtctgtgtcc
2160 cagaggaata acagctgggc attttcccca ggataacctc taaggccagc
cttgggactg 2220 ggggagagag ggaaagttct ggttcaggtc acatggggag
gcagggttgg ggctggacca 2280 ccctccccat ggctgcctgg gtctccatct
gtgtccctct atgtctcttt gtgtcgcttt 2340 cattatgtct cttggtaact
ggcttcggtt gtgtctctcc gtgtgactat tttgttctct 2400 ctctccctct
cttctctgtc ttcagtctcc atatctcccc ctctctctgt ccttctctgg 2460
tccctctcta gccagtgtgt ctcaccctgt atctctctgc caggctctgt ctctcggtct
2520 ctgtctcacc tgtgccttct ccctactgaa cacacgcacg ggatgggcct
ggggggaccc 2580 tgagaaaagg aagggctttg gctgggcgcg gtggctcaca
cctgtaatcc cagcactttg 2640 ggaggccaag gcaggtagat cacctgaggt
caggagttcg agaccagcct ggccaactgg 2700 tgaaacccca tctctactaa
aaatacaaaa aattagccag gcgtggtggc gcatgcctgt 2760 agtcccagct
actcaggagg ctgagggagg agaattgctt gaacctggga ggttgaggtt 2820
gcagtgagcc gagaccgtgc cactgcactc cagcctgggt gacagagtga gactccgcct
2880 caaaaaaaaa aaaaaaaaaa aaaaaaaaaa agaaaagaaa agaaaagaaa
aggaatcttt 2940 tatccctgat gtgtgtgggt atgagggtat gagagggccc
ctctcactcc attccttctc 3000 caggacatcc ctccactctt gggagacaca
gagaagggct ggttccagct ggagctggga 3060 ggggcaattg agggaggagg
aaggagaagg gggaaggaaa acagggtatg ggggaaagga 3120 ccctggggag
cgaagtggag gatacaacct tgggcctgca ggccaggcta cctacccact 3180
tggaaaccca cgccaaagcc gcatctacag ctgagccact ctgaggcctc ccctccccgg
3240 cggtccccac tcagctccaa agtctctctc ccttttctct cccacacttt
atcatccccc 3300 ggattcctct ctacttggtt ctcattcttc ctttgacttc
ctgcttccct ttctcattca 3360 tctgtttctc actttctgcc tggttttgtt
cttctctctc tctttctctg gcccatgtct 3420 gtttctctat gtttctgtct
tttctttctc atcctgtgta ttttcggctc accttgtttg 3480 tcactgttct
cccctctgcc ctttcattct ctctgtcctt ttaccctctt cctttttccc 3540
ttggtttctc tcagtttctg tatctgccct tcaccctctc acactgctgt ttcccaactc
3600 gttgtctgta tttttggcct gaactgtgtc ttccccaacc ctgtgttttt
ctcactgttt 3660 ctttttctct tttggagcct cctccttgct cctctgtccc
ttctctcttt ccttatcatc 3720 ctcgctcctc attcctgcgt ctgcttcctc
cccagcaaaa gcgtgatctt gctgggtcgg 3780 cacagcctgt ttcatcctga
agacacaggc caggtatttc aggtcagcca cagcttccca 3840 cacccgctct
acgatatgag cctcctgaag aatcgattcc tcaggccagg tgatgactcc 3900
agccacgacc tcatgctgct ccgcctgtca gagcctgccg agctcacgga tgctgtgaag
3960 gtcatggacc tgcccaccca ggagccagca ctggggacca cctgctacgc
ctcaggctgg 4020 ggcagcattg aaccagagga gtgtacgcct gggccagatg
gtgcagccgg gagcccagat 4080 gcctgggtct gagggaggag gggacaggac
tcctgggtct gagggaggag ggccaaggaa 4140 ccaggtgggg tccagcccac
aacagtgttt ttgcctggcc cgtagtcttg accccaaaga 4200 aacttcagtg
tgtggacctc catgttattt ccaatgacgt gtgtgcgcaa gttcaccctc 4260
agaaggtgac caagttcatg ctgtgtgctg gacgctggac agggggcaaa agcacctgct
4320 cggtgagtca tccctactcc caagatcttg aggggaaagg tgagtgggga
ccttaattct 4380 gggctggggt ctagaagcca acaaggcgtc tgcctcccct
gctccccagc tgtagccatg 4440 ccacctcccc gtgtctcatc tcattccctc
cttccctctt ctttgactcc ctcaaggcaa 4500 taggttattc ttacagcaca
actcatctgt tcctgcgttc agcacacggt tactaggcac 4560 ctgctatgca
cccagcactg ccctagagcc tgggacatag cagtgaacag acagagagca 4620
gcccctccct tctgtagccc ccaagccagt gaggggcaca ggcaggaaca gggaccacaa
4680 cacagaaaag ctggagggtg tcaggaggtg atcaggctct cggggaggga
gaaggggtgg 4740 ggagtgtgac tgggaggaga catcctgcag aaggtgggag
tgagcaaaca cctgccgcag 4800 gggaggggag ggccctgcgg cacctggggg
agcagaggga acagcatctg gccaggcctg 4860 ggaggagggg cctagagggc
gtcaggagca gagaggaggt tgcctggctg gagtgaagga 4920 tcggggcagg
gtgcgagagg gaagaaagga cccctcctgc agggcctcac ctgggccaca 4980
ggaggacact gcttttcctc tgaggagtca ggaactgtgg atggtgctgg acagaagcag
5040 gacagggcct ggctcaggtg tccagaggct gccgctggcc tccctatggg
atcagactgc 5100 agggagggag ggcagcaggg atgtggaggg agtgatgatg
gggctgacct gggggtggct 5160 ccaggcattg tccccacctg ggcccttacc
cagcctccct cacaggctcc tggccctcag 5220 tctctcccct ccactccatt
ctccacctac ccacagtggg tcattctgat caccgaactg 5280 accatgccag
ccctgccgat ggtcctccat ggctccctag tgccctggag aggaggtgtc 5340
tagtcagaga gtagtcctgg aaggtggcct ctgtgaggag ccacggggac agcatcctgc
5400 agatggtcct ggcccttgtc ccaccgacct gtctacaagg actgtcctcg
tggaccctcc 5460 cctctgcaca ggagctggac cctgaagtcc cttccctacc
ggccaggact ggagccccta 5520 cccctctgtt ggaatccctg cccaccttct
tctggaagtc ggctctggag acatttctct 5580 cttcttccaa agctgggaac
tgctatctgt tatctgcctg tccaggtctg aaagatagga 5640 ttgcccaggc
agaaactggg actgacctat ctcactctct ccctgctttt acccttaggg 5700
tgattctggg ggcccacttg tctgtaatgg tgtgcttcaa ggtatcacgt catggggcag
5760 tgaaccatgt gccctgcccg aaaggccttc cctgtacacc aaggtggtgc
attaccggaa 5820 gtggatcaag gacaccatcg tggccaaccc ctgagcaccc
ctatcaactc cctattgtag 5880 taaacttgga accttggaaa tgaccaggcc
aagactcaag cctccccagt tctactgacc 5940 tttgtcctta ggtgtgaggt
ccagggttgc taggaaaaga aatcagcaga cacaggtgta 6000 gaccagagtg
tttcttaaat ggtgtaattt tgtcctctct gtgtcctggg gaatactggc 6060
catgcctgga gacatatcac tcaatttctc tgaggacaca gataggatgg ggtgtctgtg
6120 ttatttgtgg gatacagaga tgaaagaggg gtgggatcca cactgagaga
gtggagagtg 6180 acatgtgctg gacactgtcc atgaagcact gagcagaagc
tggaggcaca acgcaccaga 6240 cactcacagc aaggatggag ctgaaaacat
aacccactct gtcctggagg cactgggaag 6300 cctagagaag gctgtgagcc
aaggagggag ggtcttcctt tggcatggga tggggatgaa 6360 gtaaggagag
ggactggacc ccctggaagc tgattcacta tggggggagg tgtattgaag 6420
tcctccagac aaccctcaga tttgatgatt tcctagtaga actcacagaa ataaagagct
6480 cttatactgt ggtttattct ggtttgttac attgacagga gacacactga
aatcagcaaa 6540 ggaaacaggc atctaagtgg ggatgtgaag aaaacaggga
aaatctttca gttgttttct 6600 cccagtgggg tgttgtggac agcacttaaa
tcacacagaa gtgatgtgtg accttgtgta 6660 tgaagtattt ccaactaagg
aagctcacct gagccttagt gtccagagtt cttattgggg 6720 gtctgtagga
taggcatggg gtactggaat agctgacctt aacttctcag acctgaggtt 6780
cccaagagtt caagcagata cagcatggcc tagagcctca gatgtacaaa aacaggcatt
6840 catcatgaat cgcactgtta gcatgaatca tctggcacgg cccaaggccc
caggtatacc 6900 aaggcacttg ggccgaatgt tccaagggat taaatgtcat
ctcccaggag ttattcaagg 6960 gtgagccctg tacttggaac gttcaggctt
tgagcagtgc agggctgctg agtcaacctt 7020 ttactgtaca ggggggtgag
ggaaagggag aagatgagga aaccgcctag ggatctggtt 7080 ctgtcttgtg
gccgagtgga ccatggggct atcccaagaa ggaggaattc 7130 12 20 DNA Homo
sapiens 12 agcattccca accctggcag 20 13 3923 DNA Homo sapiens 13
acagaagaaa tagcaagtgc cgagaagctg gcatcagaaa aacagagggg agatttgtgt
60 ggctgcagcc gagggagacc aggaagatct gcatggtggg aaggacctga
tgatacagag 120 gaattacaac acatatactt agtgtttcaa tgaacaccaa
gataaataag tgaagagcta 180 gtccgctgtg agtctcctca gtgacacagg
gctggatcac catcgacggc actttctgag 240 tactcagtgc agcaaagaaa
gactacagac atctcaatgg caggggtgag aaataagaaa 300 ggctgctgac
tttaccatct gaggccacac atctgctgaa atggagataa ttaacatcac 360
tagaaacagc aagatgacaa tataatgtct aagtagtgac atgtttttgc acatttccag
420 cccctttaaa tatccacaca cacaggaagc acaaaaggaa gcacagagat
ccctgggaga 480 aatgcccggc cgccatcttg ggtcatcgat gagcctcgcc
ctgtgcctgg tcccgcttgt 540 gagggaagga cattagaaaa tgaattgatg
tgttccttaa aggatgggca ggaaaacaga 600 tcctgttgtg gatatttatt
tgaacgggat tacagatttg aaatgaagtc acaaagtgag 660 cattaccaat
gagaggaaaa cagacgagaa aatcttgatg gcttcacaag acatgcaaca 720
aacaaaatgg aatactgtga tgacatgagg cagccaagct ggggaggaga taaccacggg
780 gcagagggtc aggattctgg ccctgctgcc taaactgtgc gttcataacc
aaatcatttc 840 atatttctaa ccctcaaaac aaagctgttg taatatctga
tctctacggt tccttctggg 900 cccaacattc tccatatatc cagccacact
catttttaat atttagttcc cagatctgta 960 ctgtgacctt tctacactgt
agaataacat tactcatttt gttcaaagac ccttcgtgtt 1020 gctgcctaat
atgtagctga ctgtttttcc taaggagtgt tctggcccag gggatctgtg 1080
aacaggctgg gaagcatctc aagatctttc cagggttata cttactagca cacagcatga
1140 tcattacgga gtgaattatc taatcaacat catcctcagt gtctttgccc
atactgaaat 1200 tcatttccca cttttgtgcc cattctcaag acctcaaaat
gtcattccat taatatcaca 1260 ggattaactt ttttttttaa cctggaagaa
ttcaatgtta catgcagcta tgggaattta 1320 attacatatt ttgttttcca
gtgcaaagat gactaagtcc tttatccctc ccctttgttt 1380 gatttttttt
ccagtataaa gttaaaatgc ttagccttgt actgaggctg tatacagcac 1440
agcctctccc catccctcca gccttatctg tcatcaccat caacccctcc cataccacct
1500 aaacaaaatc taacttgtaa ttccttgaac atgtcaggac atacattatt
ccttctgcct 1560 gagaagctct tccttgtctc ttaaatctag aatgatgtaa
agttttgaat aagttgacta 1620 tcttacttca tgcaaagaag ggacacatat
gagattcatc atcacatgag acagcaaata 1680 ctaaaagtgt aatttgatta
taagagttta gataaatata tgaaatgcaa gagccacaga 1740 gggaatgttt
atggggcacg tttgtaagcc tgggatgtga agcaaaggca gggaacctca 1800
tagtatctta tataatatac ttcatttctc tatctctatc acaatatcca acaagctttt
1860 cacagaattc atgcagtgca aatccccaaa ggtaaccttt atccatttca
tggtgagtgc 1920 gctttagaat tttggcaaat catactggtc acttatctca
actttgagat gtgtttgtcc 1980 ttgtagttaa ttgaaagaaa tagggcactc
ttgtgagcca ctttagggtt cactcctggc 2040 aataaagaat ttacaaagag
ctactcagga ccagttgtta agagctctgt gtgtgtgtgt 2100 gtgtgtgtgt
gagtgtacat gccaaagtgt gcctctctct cttgacccat tatttcagac 2160
ttaaaacaag catgttttca aatggcacta tgagctgcca atgatgtatc accaccatat
2220 ctcattattc tccagtaaat gtgataataa tgtcatctgt taacataaaa
aaagtttgac 2280 ttcacaaaag cagctggaaa tggacaacca caatatgcat
aaatctaact cctaccatca 2340 gctacacact gcttgacata tattgttaga
agcacctcgc atttgtgggt tctcttaagc 2400 aaaatacttg cattaggtct
cagctggggc tgtgcatcag gcggtttgag aaatattcaa 2460 ttctcagcag
aagccagaat ttgaattccc tcatctttta ggaatcattt accaggtttg 2520
gagaggattc agacagctca ggtgctttca ctaatgtctc tgaacttctg tccctctttg
2580 tgttcatgga tagtccaata aataatgtta tctttgaact gatgctcata
ggagagaata 2640 taagaactct gagtgatatc aacattaggg attcaaagaa
atattagatt taagctcaca 2700 ctggtcaaaa ggaaccaaga tacaaagaac
tctgagctgt catcgtcccc atctctgtga 2760 gccacaacca acagcaggac
ccaacgcatg tctgagatcc ttaaatcaag gaaaccagtg 2820 tcatgagttg
aattctccta ttatggatgc tagcttctgg ccatctctgg ctctcctctt 2880
gacacatatt agcttctagc ctttgcttcc acgactttta tcttttctcc aacacatcgc
2940 ttaccaatcc tctctctgct ctgttgcttt ggacttcccc acaagaattt
caacgactct 3000 caagtctttt cttccatccc caccactaac ctgaatgcct
agacccttat ttttattaat 3060 ttccaataga tgctgcctat gggctatatt
gctttagatg aacattagat atttaaagct 3120 caagaggttc aaaatccaac
tcattatctt ctctttcttt cacctccctg ctcctctccc 3180 tatattactg
attgcactga acagcatggt ccccaatgta gccatgcaaa tgagaaaccc 3240
agtggctcct tgtggtacat gcatgcaaga ctgctgaagc cagaaggatg actgattacg
3300 cctcatgggt ggaggggacc actcctgggc cttcgtgatt gtcaggagca
agacctgaga 3360 tgctccctgc cttcagtgtc ctctgcatct cccctttcta
atgaagatcc atagaatttg 3420 ctacatttga gaattccaat taggaactca
catgttttat ctgccctatc aattttttaa 3480 acttgctgaa aattaagttt
tttcaaaatc tgtccttgta aattactttt tcttacagtg 3540 tcttggcata
ctatatcaac tttgattctt tgttacaact tttcttactc ttttatcacc 3600
aaagtggctt ttattctctt tattattatt attttctttt actactatat tacgttgtta
3660 ttattttgtt ctctatagta tcaatttatt tgatttagtt tcaatttatt
tttattgctg 3720 acttttaaaa taagtgattc ggggggtggg agaacagggg
agggagagca ttaggacaaa 3780 tacctaatgc atgtgggact taaaacctag
atgatgggtt gataggtgca gcaaaccact 3840 atggcacacg tatacctgtg
taacaaacct acacattctg cacatgtatc ccagaacgta 3900 aagtaaaatt
taaaaaaaag tga 3923
* * * * *