Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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A METHOD FOR DETECTION OF PROSTATE SPECIFIC ANTIGEN USED
IN MONITORING AND DIAGNOSIS OF PROSTATE CANCER
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No.
60/0~5,765, filed April l6, 1996.
BACKGROUND OF THE INVENTION
Cancer of the prostate is the most prevalent mqlignqllty in adult males,
eYel~ ng skin cancer, and is an increasingly prevalent health plubl~ in the United
States. In 1994, it was c,li...~t~c~ that 38,000 deaths resulted from this disease,
inrli~qting that prostate cancer is second only to lung cancer as the most COI~ II cause
of death in the sarne populatiûn. If rliqgnosed early, when the cancer is still confined
to the plo~laLe, the chances of cure is ~i~nifirqntly higher. Accordingly, there is a great
15 need for sensitive m~tho-1s for the detection of organ-confine prostate cancer.
Recently, polylll.,.ase chain reaction (PCR) and vqriqtionc thereof, have been
used to detect mqti~nqnt cells for ~lia~osic and monit- rin~ of various mqlignqrlrie5
For e~rq-mple, reverse~ s~ t~se PCR (RT-PCR) is a pO~.rul technique wh~ehcan
be used to detect the presence of a specific mRNA populatiûn in a complex mLlcture ûf
20 thousands of other mRNA species. In RT-PCR, an mRNA species is first reverse
tr,-lcrribecl to co...pl~ "r.,t~"y DNA (cDNA) with use of the enzyme reverse
transcli~se; the cDNA is then amplified as in a standard PCR reaction. RT-PCR can
thus reveal by AmplifirAtiQn the presence of a sing}e species of mRNA. Accordingly, if
the mRNA is highly specific for the cell that produces it, RT-PCR can be used to25 identify the presence of a specific type of cell.
Prostate-specific antigen (PSA), a 33-kDa protein, was originAlly thought to be
produced extensively by prostatic epith~.liAl cells and thus was used as a tumor marker
to aid in the detection and monitoring of prostate cancer. More recent studies indicate
that PSA is not specific for prostatic tissue. For example, DiAmAn-lic and Yu reported
30 the presence of PSA in 30% of female breast tumors, more rarely in other tumors, in
. . ~ , ,.. ~ .. .
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norrnal breast tissue and in biological fluids such as breast mil~. Clin. Chem. 1995,
41(2):177-179. PSA has also been ~letected in enclom~t~ial tissue.
RT-PCR of rnRNA encoding for PSA has been used in the identification of
prostate cancer cells. For exarnple, Deguchi et al. detected PSA rnRNA in regional
lymph nodes from patients with prostate cancer which had m~t~ct~ci7ed to the
lymphatic system. Cancer Res. 1993, 53:53504. RT-PCR methods for detection of
prostate cancer cells in the general circulation of patients with advanced prostate
cancer have also been disclosed. Moreno et al. Cancer ~es. 1992, 53:6110-2; Jaakkola
et al. Clin. Chem. 1995, 41(2):182-186. However, patients with early prostate cancer
and/or well di~r.,.e~ t~l or localized disease were found negative for PSA mRNA by
PCR. Accordingly, it wa~s con~ 1P~l that RT-PCR for PSA mRNA would not be a
useful tool for either the ~ gnosic or monitoring of prostate cancer. Di~m~nt1is, E.P.
and Yu, H. Clin. Chem. 1995, 41(2):177-179.
The present invention provides a new RT-PCR m~tho~ with novel primers for
the monitoring and diagnosis patients with prostate cancer. This method is d~ci~gnPd
to det~Tnine the presence of rnRNA for PSA in the bloodstrearn thereby providingevidence that p~ L~ cells are circ~ tin,, in the blood and confirming a ~ gnocic of
cancer. Circulating prostate cells also in~ e that the patient is at risk of secondary
tumor or bone mPt~ct~tic anchoring. Sarnples taken after prost~tectomy or radiation
treatment which are reactive indicate the risk that the prostate cancer had spread and
that surgery/radiation was not efficacious.
SUMMAI~Y OF THE INVENTION
An object of the present invention is to provide primers for use in an RT-PCR
method for the detection of prostatic specific antigen for the monitoring and diagnosis
of patients with prostate cancer.
Another object of the present invention is to provide a method for monitoring
the progression of prostate cancer which comprises dP-tecting prostate specific antigen
in patient with an RT-PCR method which uses these primers.
Another object of the present invention is to provide a kit for monitoring, and
diagnosis of prostate cancer which comprises these primers.
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BRIEF DESCRIPTION OF THE FIGURES
Figure 1 provides the human DNA for prostate specific antigen (PSA) (SEQ
ID NOS: 1, 8, 9 and 10). Introns are abbreviated for clarity. Exons are indicated in
~ 5 bold. Primers previously disclosed in the art are indic~ted by underlining. The
primers of the present invention, namely DR PSA-up (SEQ ID NO: 2), DR PSA-
Down (SEQ ID NO: 3) and DR 12 mer (SEQ ID NO: 4), are in~ te~ by underlined
italics in a larger font size.
DETAILED DESCRIPrION OF THE INVENTION
Expression of mRNA specific for a certain tissue in peripheral blood is
believed to in(1irat~ the presence of circ~ ting cancer cells and rnet~t~tiC spread of a
tumor origin~ting from this tissue. The detection of PSA mRNA in peripheral blood
has been associated with m~t~ct~tic prostate cancer, however, PSA mRNA has also
been detected in the blood of patients with clinically confined prostate cancer
Accordingly, clinical methods of staging prostate cancer often understage patients and
underestim~t~. the risk of m~ t~t~ic
A new method for the detection of ~r~sLaLe; specific antigen by reverse-
transcriptase polymerase chain reaction has been developed which can be used in the
monitoring of prostate cancer in a patient. Primers which are used in the method of the
present invention include DR PSA-Up: 5'-GTTGTCTTCCI CACCCTGTCCG-3'
(SEQ ID NO: 2), DR PSA-Down: 5'-TCCAGCACACAGCATGAACTTG-3' (SEQ
ID NO: 3), and DR 12 mer: 5'-GAATCACCCGAG-3' (SEQ ID NO: 4). The method
of the present invention comprises isolation and washing of buffy coat cells, isolation
of total RNA from buffy coat cells, reverse transcription (RT) reaction for PSA, PCR
for PSA, and detection of the absence or presence of PSA product on a DNA
sequencer. The method may further comprise RT reaction and PCR for a
house~eping gene Beta-2-microglobulin (B2G: Israeli et al. Cancer Research 1994
54:6306-6310) to monitor quality of the isolated RNA.
In this method, Vacutainer Cell P~ ~ation Tubes (CPT) (Becton Dickinson
Vacutainer Systems Cat. #362761 Sodium Citrate anticoagulant, 8 ml draw, San Jose,
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CA) are used to collect blood samples from patients suspected of having prostatecancer. These samples are then centrifuged in accordance with the m~nllf~tnrer'sinstructions to separate the buffy coat from other blood components. Buffy coat cells
are then washed with PBS and pelleted by centrifugation. The pelleted cells are taken
5 up in a cell Iysis buffer. Cells are homogenized by passage over a Qiagen QIA
shredder rnicrospin column. The run-through is then passed over a Qiagen RNeasy
column to bind RNA. The sample is washed to remove DNA and other components,
and the RNA is eluted with DEPC-treated water. The eluted sample RNA is then used
as the substrate in the RT reactions. The RT reaction primer for PSA reactions is DR
10 12 mer (SEQ ID NO: 4) which is specific for the PSA target. An aliquot of the RT
reaction is then used for the P(:R reaction for PSA, using primers DR PSA Up (SEQ
ID NO: 2) and DR PSA Down (SEQ ID NO: 3). In a ~lGfe.l~d embo~im~nt wherein
the method further comrrices RT reaction and PCR for B2G to monitor quality of the
isolated RNA, the RT reaction primer is 5'-AG~ l-l~AGTGC-3' (SEQ ID NO: 5)
15 and the PCR reaction primers are 5'-AGCAGAGAATGGAAAGTCAAA-3' (SEQ ID
NO: 6) and 5'-TGT TGATGT TGGATAAGAGAAT-3' (SEQ ID NO: 7). The sizes of
the products from each reaction for each patient are compared on the ABI 373 DNAAnalyzer with the ABI 672 GENESCAN software. This combination provides the
sensitivity required to deterrnin~ the presence of as high a dilution as 1 or fewer
20 LNCaP (PSA-producing prostate cancer tissue culture) cells per one million white
blood cells (WBCs). The average 8 rnl blood sample will contain approximately 40million WBCs. The ABI 672 GENESCAN software can dete~Tnin~ the size in bases of
each product, allowing definitive iclentifi~tion of each product.
The specificity of the method of the present invention in detecting prostate
25 cancer was deterrnined. PSA levels in blood samples from normal patients and
patients having benign prostatic hypertrophy (BPH), prostatitis, and varying stages of
prostate cancer were measured. Results from these e~rerim~ntc are shown in the
following Table I .
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~PR~VATE }Disease Total # of # of Patients r~ilive % S~ilivil~
State Patientc for PSA
Norrnal 84 0 100%
BPH 16 0
Prostatitis 5 o
Prostate Cancer Status
Progressive 14 11 78.6
Stable ~ 5 years 4 6 66.6
Stable > 5 years 4 0
StageB1 orB2 4 0
Normal donors included males under the age of 35 and females. BPH s~mples
included patients diagnosed with BPH but not cancer. Prostate cancer status was
5 provided by the patient's physician.
As is demonstrated by these results, the method of the present invention is
specific for determining met~ct~ti~ ploslale cancer, in particular prostate cancer in the
progressive stage and that which has been stable for less than 5 years. By
"progressive" it is meant cancer with identified metastases to the bone or other sites,
10 with the mP.t~ct~ses enlarged andlor more numerous than in the last çx~min~tion.
Patients who have been stable less than S years hi.ctori~lly suffer approximately a
66% relapse rate before the end of 5 years. The results in Table 1 are consistent with
this relapse rate. Tests with the assay of the present invention predict that the 2 out of
6 nonreactive patients in this group will remain stable for over 5 years while the 4 out
15 of 6 reactive patients have a high probability of relapse. Samples from patients having
stage B cancer, or organ-confined cancer, which is not metastatic were nonreactive in
the method of the present invention. Further, stable patients, mto~ning those with
m~t~c~ses in the past but no further progression in 5 or more years, who are
considered cured, were also nonreactive.
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Recent studies indicate that circulating prostate cells can also result from
proSt~tpctQmy operations. Oefelein et al. J. Urol. 1996 1~:238-242. If cancerous,
these circul~ing cells caused by the surgery could result in metastases. The method of
the present invention can be used in monitoring these surgical procedures to ensure
that a patient is nonreactive prior to surgery as well as after the surgery.
The present invention also provides kits for monitoring of progression of
prostate cancer which comprise the DR PSA-Up (SEQ ID NO: 2), DR PSA-Down
(SEQ ID NO: 3), and DR 12 mer (SEQ ID NO: 4) primers. In a preferred
embo-1imPnt, these kits also compri.ce primers for RT and PCR detection of B2G such
as SEQ ID NOs: 5, 6 and 7. Further, kits of the present invention may comprise tubes
for collection of blood s~mrles which are capable of separating the buffy coat from
other blood components such as Va~ut~inPr Cell Ple~Lion Tubes. Kits of the
present invention may also comprise a means for isolating RNA. For example, in one
emboflimP-nt the kit may comprice a cell lysis buffer, Qiagen QIA shredder microspin
colurnns (Cat. #79655) for cell homogenization and Qiagen RNeasy columns (Cat.
#74106) for binding of the RNA. The kits may also contain any of the following
reagents including, but not lirnited to, Beta-Mercaptoethanol (BME), Diethyl
Pyrocarbonate (DEPC), SuperScript II RNase H- Reverse Transcriptase (Gibco BRL,
G~ith~rcburg, MD), ethanol (200 Proof), 100 mM dNTPs, AmpliTaq D~A
Polymerase (Perkin Elmer Cat. #N808-0153), 6% (6% T, 5%C) den~tllnn~ acrylamide
gel Gibco/BRL 6% Sequencing Solution (Gel-Mix 6; Cat. #5543UA), 10 %
ammonium persulfate,
Denaturing loading buffer corlt~inin~ Blue dextran (50 mg/ml) and 200 ml formamide
and 20 ml 100 mM EDTA, lX TBE for Sequencer Gibco/BRL UltraPure Gel-Mix
running mate (Cat. #15546-013) and Brij 35 (Sigma Chemical Co. St. Louis, MO, Cat.
#430AG-6). Standard and controls may also be provided in the kits of the presentinvention. Examples include, but are not limited to Genesc~n ROX-2500 standard
(Perkin Elmer/A13I Cat. #401100), RNA from ATCC cell lines CRL-1435 PC-3
(prostate adenocarcinoma, human) or HlB-22 MCF7 (breast adenocarcinoma, pleural
effusion, human), and RNA from ATCC cell line CRL-1740 LNCaP (m~t~ct~tic
prostate adenocarcinoma, human).
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The following nonlimiting exarnples are provided to further illustrate the
present invention.
EXAMPLES
S EXAMPLE 1: Preparation of Reagents
Reagents are prepared in ess~nti~lly RNAse-free, sterile, disposable
plasticware, in glassware baked at 180~C for at least 8 hours, or in polypropylene
plasticware rinsed with chloroform.
Solutions used for RNA and reverse transcription work are prepared using
RNAse-free glassware, autoclaved water, and chemicals reserved for RNA work
handled with baked spat~ c Water for solutions is treated with 0.1% DEPC for at
least 12 hours at 37~C and then heated to 100~C for 15 minlltes or autoclaved for 15
minl-t~s at 15 lb/sq. inch on liquid cycle.
Phosphate buffered saline (PBS) used in this method is prcp~d by dissolving
8 grams of NaCI, 0.2 grams of KCI, 1.44 grams of Na2HPO4, and 0.24 grams of
KH2PO4 in 800 ml of distilled H20. The pH of the buffer is adjusted to 7.4 with HCI.
The volume is then adjusted to l liter with H20. Prior to use, the buffer is Ste~ ili7~d by
autoclaving for 20 minutPc at 15 Ib/sq. inch on liquid cycle and stored at room
te~ eldlure.
Lysis Buffer (BME) is prepared from 100 ~1 of BME per 10 ml of QIAGEN
RNeasy kit Lysis Buffer RLT. The resulting Lysis Buffer/BME is stable for 1 month.
Wash Buffer RPE is ple~cd for use by adding 4 volumes of 96-100% ethanol
to the concentrate supplied by Qiagen.
Seventy percent Ethanol is prepared by adding 700 ml of 200 proof Ethanol to
300 ml DEPC-treated water. The solution is stored at room temperature.
FY~rnrle 2: White Blood Cell Isolation
Blood sarnples are obtained in Vacutainer CPT tubes with sodium citrate. The
samples are then centrifuged at room temperature (20 - 25~C) in a horizontal swing-out
head rotor at 1500 to 1800 RCF (Relative Centrifugal Force) for 30 minu~s After
centrifugation, the buffy coat appears as a whitish layer just under the plasma layer.
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Approximately half of the plasma is aspirated without disturbing the cell layer. The
cell layer is then collected with a sterile pipette and transferred to a I5 rnl conical
centrifuge tube.
PBS is added to the conical tubes to bring the volume to 15 ml. The tubes are
then capped and the cells and PBS mixed by inverting the tubes 5 times. The tubes are
then centrifuged for 15 minl-tes at 300 RCF and the supern:lt.~nt is aspirated without
disturbing the cell pellet. PBS is again added to the cells to bring the volume to 10 ml.
The tubes are capped and the cells and PBS are mixed by inverting the tubes 5 times.
The tubes are then centrifuged for 10 minute,c at 300 RCF. As much sup~rn~t~nt as
possible is aspirated without disturbing the cell pellet. Lysis Buffer/BME (400 111) is
added to the pellet and the cells are ~ e.~ d by gentle vortexing. At this stage,
samples may be frozen at -70~C or used immPrli~t~ly for RNA isolation.
Example 3: RNA T.col~tinn
The cells/Lysis Buffer/BME sUcpen.cion is pipetted directly onto a QIAGEN
QIA shredder column and centrifuged at full speed for 1 nunute in a microcentrifuge.
The time of centrifugation may be ~~~ntled if liquid remains above the colurnn. One
volume (400 ~Ll) of 70% ethanol is added to the homogenized Iysate and mixed. The
sample is then applied to an RNeasy spin colurnn and centrifuged for 15 seconds at
8000 x g (10,000 rpm). Flowthrough is discarded. Wash Buffer RWl (700 ,ul) is then
pipetted onto the spin column. The columns are again centrifuged and the flowthrough
is discarded. Following this wash, the spin column is placed in a new 2 ml collection
tube. Wash Buffer RPE (500 ~11) is pipetted onto the spin colurnn and centrifuge as
above. The flowthrough is discarded. A second aliquot of Wash Buffer RPE is
pipetted onto the spin colurnn and centrifuged for 2 min~ltec at full speed to dry the
RNeasy spin colurnn. This 2 minute spin assures that no residual ethanol will becarried over during elution. The spin column is then transferred to a new 1.5 mlcollection tube and the sarnple RNA is eluted with 50 ~1 of DEPC-treated water
pipetted directly onto the spin column membrane followed by centrifugation for 60
seconds at 8000 x g. Sample RNA may be used immt~ tely in the 3~T reaction, or
stored at -70~C until further use.
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Example 4: Determining the RNA co~c~ntration
The RNA concentration in each sample is determined by W
spectrophotometry. The baseline absorbance of 75 lul of distilled water is firstdetermined at 260, 280, and 320 nm. A five microliter sample of RNA is then added
and mixed. The sarnple absorbance is then determined at 260, 280, and 320 nm. The
neat RNA concentration is c~lcul~tecl by:
a) subtracting the A320 from the A260 to obtain the absorbance due to RNA;
b) multiplying by the dilution factor (80 ,ul5 ,ul); and
c) multiplying by the conversion factor (0.04 mg/ rnl).
Example 5: Sample Reverse Tr~ tion
Two microliters of 50 ~M PSA RT primer (SEQ ID NO: 4) are pipetted into
each PCR reaction tubes. Two microliters of 50 ~LM B2G RT primer (SEQ ID NO: 5
are pipetted into separate PCR reaction tubes. Tubes may be prepared in advance and
stored at -20~C.
RT Reaction Mix is plc~al~d just prior to use in the following manner: The
number of sample reactions (PSA and B2G) to be run is first determined as variable X.
The following volumes of reagents are then rnixed: 4(X+1) ~Ll 5X First Strand Buffer;
2(X+1) ~Ll 0.1 M DTT; 2(X+1) ~11 20 mM dNTP solution; l(X+1) ~IL SuperScript II
reverse transcriptase. The solution is then vortexed to ensure homogeneity.
Two tubes per RNA sample are prepared, one tube for each reaction: PSA and
B2G. Ten microliters of sample RNA are added to each reaction tube. ~11 RT
reaction tubes are then placed in a thermal cycler programmed for 70~C for 10
minutes. Within the last minute of the program, the thermal cycler is set to PAUSE
and each tube is placed on ice. Ten microliters of RT Reaction Mix are then added to
each tube and the following program is run: 25~C for 5 minute~; 42~C for 15 minllte~;
97~C for 15 seconds; followed by 4~C for at least 5 minutes.
Example 6: S~n~ple Amplification
PSA PCR Reaction Mix is prepared in the following manner.
The number of PSA sample reactions to be run is det~rrnined as variable X. The
following volumes of reagents are then mixed:
g
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31.95(X+1) ~ O; 4(X+l) ~LI 10X PCR Buffer II; (X+1) ~11 20 mM dNTP; 0 8(X+1)
~LI 25 rnM MgCl2; 2(X~ Ll PSA primers (SEQ ID NOs: 2 and 3); and 0 25(X+1) 111
Taq enzyme.
B2G PCR Reaction Mix is p.epa~d in the following manner:
5 The number of B2G sample reactions to be run is determined as variable X. The
following volumes of reagents are then mixed: 33.15(X+l) ,ul H2O; 4(X+1) ~LI 10XPCR Buffer II; (X+1) ~ll 20 mM dNTP; 0.8(X+1) ~125 rnM MgCl2; 0.8(X+l) ~L} B2G
primers (SEQ ID NOs: 6 and 7); and 0.25(~+1) ,ul Taq enzyme.
Two tubes per RNA sample are prepared, one tube for each reaction: PSA and
10 B2G. Forty microliters of the a~loyliate reaction rnixture are pipetted into each of the
tubes Ten rnicroliters of the respective sarnple RNA or control RNA Reverse
Transcription reaction are pipeKed into each tube. For the negative (reaction) control,
10 rnl of water are used. The tubes are spun for 10 seconds in an Eppendorf
rnicrocentrifuge to ensure all liquid is mixed together at the bottom of the tubes. The
15 tubes are then placed in the Perkin Elmer 9600 Therrnocycler and cycled as follows:
Cycle Type Temp Time # of Cycles
step cycle 95~C 15 sec 30
58~C 15 sec
72~C 45sec
hold 72~C 3 minllteS
hold 4~C indefinite
F,Y5~.nlJle 7~ nrle Detection
Two 6 cm well-to-read plates (one notched, one plain) are prepared for use in
the following manner. Both sides of each plate are rinsed with distilled water. One
side only of each plate is then selected as the inside (gel side) of the plate. This inside
is then rinsed with methanol followed by 0.5% Brij.
The methanol rinse is then repeated until the plates are streak free.
Spacers are then placed on the edge of the plain plate, parallel to the short side
of the plates. The notched plate is placed on top of the plain plate, with the insides of
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the plates facing each other. The plates are then stood on their bottom edge and the
spacers lined up so they are level with the bottom and sides of the gel. They are gently
laid down flat with the plain plate on the bottom and clamped with 6 binder clips.
Seventy-five milliliter.c of ammonium persulfate is added to 20 rnl of Gel-Mix
5 6 and swirled gently to mix. The solution is then poured between the sequencing
plates. A sample comb is inserted and allowed to polymerize 1-2 hours.
After polymPn7~tion is complete, the clamps are removed and the outside of
the plates are cleaned with 11i.ctille~1 water, being careful to remove all acrylarnide from
the outside surfaces of the plates. The plates are then inserted into a 373 sequencer and
10 a plate check is pc,ro~ ed in accordance with the m~nllf~hlrer~s directions to check
for in~elrele,lce. The top and bottom buffer reservoirs are filled with 700 ml and 300
ml of lX TBE, I.,;,pecLively. The sample comb is removed from between the plates,
and acrylarnide and urea are washed from the wells with lX l'BE.
The gel is prerun for 5 minutes at 28 watts.
Two rnicroliters of PCR reaction are pipetted into a 0.2 ml thin-walled PCR
reaction tube. Den~hlring sarnple buffer (2X; 2.5 ml) and 0.3 ml of Gent sc~n
ROX-2500 standard are then added. The tubes are placed in a PE 9600 Therrnocycler
and run at 94~C for 10 minlltes. Within the last rninute of the prograrn, the thermal
cycler is set to PAUSE and each tube is placed on ice. Four rnicroliters of cooled
20 sample are then loaded onto the gel. The ABI sequencer is set to run for 3.5 hours at
28 watts, 600 volts, 40 milli~mps and the Gçnçsc~n data collection to collect for 3
hours.
Expected values for each of the primer set amplifications are as follows:
MarkerSize (bp) Observed Range
B2G 537 529 - 540
PSA 586 585 - 588
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SEQUENCE LISTING
~l) GENERAL INFORMATION
~i) APPLICANT: SmithKline Beecham Corporation
(ii) TITLE OF THE INVENTION: A Method for Detection of
Prostate Specific Antigen used in Monitoring and Diagnosis
of Prostate Cancer
(iii) NUMBER OF SEQUENCES: l0
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporatio~
(B) STREET: 709 Swedeland Road
(C) CITY: King of Prussia
(D~ STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19406-0939
(v) COMPUTER READABLE FORM:
(A~ ~EDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
~A~ APPLICATION NUMBER: 60/015,765
(B) FILING DATE: l6-APR-l996
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(viii) ATTORNEY/AGENT TNFOR~ATION:
(A) NAME: Han, William T
(B~ REGISTRATION NUMBER: ~219
~C) REFERENCE/DOCKET NUMBER: P50473
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5219
0 (B) TELEFAX: 610-270-5090
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 92 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CCAAGCTTAC CACCTGCACC CGGAGAGCTG TGTCACCATG TGGGTCCCGG ~ll~l~llCCT 60
CACCCTGTCC GTGACGTGGA TTGGTGAGAG GG 92
(2) INFORMATION FOR SEQ ID NO:2:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
.... ... . .
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(xi) SEQUENCE DESCRIP~ION: SEQ ID NO:2:
G~ llCC TCACCCTGTC CG 22
(2) INFORNATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) ~uu~ DESCRIPTION: SEQ ID NO:3:
TCCAGCACAC AGCATGAACT TG 22
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) ~:Q~kNC~ DESCRIPTION: SEQ ID NO:4:
GAATCACCCG AG 12
(2) INFORMATION FOR SEQ ID NO:5:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
S AGCTTTGAGT GC 12
(2) INFORMATION FOR SEQ ID NO:6:
li) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) ~Qu~ DESCRIPTION: SEQ ID NO:6:
AGCAGAGAAT GGAAAGTCAA A 2l
( 2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
lc) sTR~NnFn~cs single
~D) TOPOLOGY: linear
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
TGTTGATGTT GGATAAGAGA AT 22
(2) INFORMATION FOR SEQ ID NO:8:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 208 base pairs
(B) TYPE: nucleic acid
- 15-
CA 022~ 7 1998-10-13
W O 97/39139 PCT~US97/06497
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
s
txi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
TGATCTAGCA CCCCCTCTGC AGGCGCTGCG CCCCTCATCC ~ lCGGAT TGTGGGAGGC 60
TGGGAGTGCG AGAAGCATTC CCAACCCTGG CAGGTGCTTG TGGCCTCTCG TGGCAGGGCA l20
GTCTGCGGCG ~lGll~l'GGT GCACCCCCAG TGGGTCCTCA CAGCTGCCCA CTGCATCAGG l80
AAGTGAGTAG GGGCCTGGGG TCTGGGGA 208
~2) INFORMATION FOR SEQ ID NO:9:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 639 ~ase pairs
~B) TYPE: nucleic acid
~C) STRANDEDNESS: single
~D) TOPOLOGY: linear
~ii) MOLECULE TYPE: cDNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
ATCATCCTCG CTCCTCATTC CTGCGTCTGC TTCCTCCCCA GCAAAAGCGT GATCTTGCTG 60
GGTCGGCACA GC~l~lllCA TCCTGAAGAC ACAGGCCAGG TATTTCAGGT CAGCCACAGC 120
TTCCCACACC CGCTCTACGA TATGAGCCTC CTGAAGAATC GATTCCTCAG GCCAGGTGAT l80
GACTCCAGCC ACGACCTCAT GCTGCTCCGC CTGTCAGAGC CTGCCGAGCT CACGGATGCT 240
GTGAAGGTCA TGGACCTGCC CACCCAGGAG CCAGCACTGG GGACCACCTG CTACGCCTCA 300
GGCTGGGGCA GCATTGAACC AGAGGAGTGT ACGCCTGGGC CAGATGGTGC AGCCGGGAGC 360
CCAGATGCCT GGGTCTGAGG GAGGAGGGGA CAGGACTCCT GGGTCTGAGG GAGGAGGGCC 420
AAGGAACCAG GTGGGGTCCA GCCCACAACA ~lGlllllGC CTGGCCCGTA GTCTTGACCC 480
CAAAGAAACT TCA~l~l~lG GACCTCCATG TTATTTCCAA TGAC~ ~l GCGCAAGTTC 540
ACCCTCAGAA GGTGACCAAG TTCATGCTGT GTGCTGGACG CTGGACAGGG GGCAAAAGCA 600
CCTGCTCGGT GAGTCATCCC TACTCCCAAG ATCTTGAGG 639
-16-
CA 022~ 7 l998-l0-l3
W O 97/39139 PCT~US97/06497
~2~ INFOR~ATION FOR SEQ ID NO 10
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH 491 base pairs
(B) TYPE nucleic acid
(C) STRANDEDNESS single
(D) TOPOLOGY linear
(ii) MOLECULE TYPE cDNA
(xi) SEQUENCE DESCRIPTION SEQ ID NO 10
ATCTCACTCT CTCCCTGCTT TTACCCTTAG GGTGATTCTG GGGGCCCACT ~ AAT 60
G~l~lG~lliC AAGGTATCAC GTCATGGGGC AGTGAACCAT GTGCCCTGCC CGAAAGGCCT 120
TCC-~ ACA CCAAGGTGGT GCATTACCGG AAGTGGATCA AGGACACCAT CGTGGCCAAC 180
CCCTGAGCAC CCCTATCAAG TCCCTATTGT AGTAAACTTG GAACCTTGGA AATGACCAGG 240
CCAAGACTCA AGC~lCCCCA GTTCTACTGA C~lll~lCCT TAGGTGTGAG GTCCAGGGTT 300
GCTAGGAAAA GAAATCAGCA GACACAGGTG TAGACCAGAG 'l'~'l"l"l'~"l"l'AA ATGGTGTAAT 360
TTTGTCCTCT ~~ C-lG GGGAATACTG GCCATGCCTG GAGACATATC ACTCAATTTC 420
TCTGAGGACA CAGTTAGGAT GGGGl~l~lG TGTTATTTGT GGGATACAGA GATGAAAGAG 480
GGGTGGGATC C 491
