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US 201001 24746A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0124746A1 Liew (43) Pub. Date: May 20, 2010 (54) METHOD FOR THE DETECTION OF GENE (60) Provisional application No. 60/115,125, filed on Jan. TRANSCRIPTS IN BLOOD AND USES 6, 1999. THEREOF (75) Inventor: Choong-Chin Liew, Toronto (CA) Publication Classification Correspondence Address: (51) 5,/68 (2006.01) EDWARDS ANGELL PALMER & DODGE LLP P.O. BOX SS874 (52) U.S. Cl. ............................................................ 435/6 BOSTON, MA 02205 (US) (73) Assignee: GeneNews, Inc, (57) ABSTRACT (21) Appl. No.: 12/587,382 The present invention is directed to detection and measure ment of gene transcripts in blood. Specifically provided is a (22) Filed: Oct. 5, 2009 RT-PCR analysis performed on a drop of blood for detecting, O O diagnosing and monitoring diseases using tissue-specific Related U.S. Application Data primers. The present invention also describes methods by (63) Continuation of application No. 10/268,730, filed on which delineation of the sequence and/or quantitation of the Oct. 9, 2002, now Pat. No. 7,598,031, which is a con expression levels of disease-associated genes allows for an tinuation of application No. 09/477,148, filed on Jan. immediate and accurate diagnostic/prognostic test for disease 4, 2000, now abandoned. or to assess the effect of a particular treatment regimen. Patent Application Publication May 20, 2010 Sheet 1 of 7 US 2010/O124746A1 3) RT PCR RT Figure 1 Patent Application Publication May 20, 2010 Sheet 2 of 7 US 2010/O124746A1 Zerr?6?EI Patent Application Publication May 20, 2010 Sheet 3 of 7 US 2010/O124746A1 Figure 3 Patent Application Publication May 20, 2010 Sheet 4 of 7 US 2010/O124746A1 Figure 4 Patent Application Publication May 20, 2010 Sheet 5 of 7 US 2010/0124746A1 Stardardized levels of insulin gere expressed in a drop of El old, A 5 1.0 0.5 0.0 lir-ul-lo -- 22:23 inserosases. a 2. Sa 3 Normal Asymptomatic Diabetic Subjects Standardized levels of ZFP gene expressed in a drop of blood. : B : S. 24 B x 1.2 s 0.6 0.0 5 s Olabatic Subjects Standardized levels of listill gate expressed ir each fractionisted call fron who blood, O.9 - 0.5 (),3 (). A. as: G.R. CD 3+ C 9. ONO Fractionated cell type Figure 5 Patent Application Publication May 20, 2010 Sheet 6 of 7 US 2010/O124746A1 Figure 6 Patent Application Publication May 20, 2010 Sheet 7 of 7 US 2010/O124746A1 | pool?uelunH US 2010/01 24746 A1 May 20, 2010 METHOD FOR THE DETECTION OF GENE and infectious disease in the human body. The process TRANSCRIPTS IN BLOOD AND USES described herein requires a simple blood sample and is, there THEREOF fore, non-invasive compared to conventional practices used to detect tissue specific disease, Such as biopsies. CROSS-REFERENCE TO RELATED 0008. One object of the present invention is to provide a APPLICATION non-invasive method for the diagnosis, prognosis and moni 0001. This application is a continuation of application Ser. toring of genetic and infectious disease in humans and ani No. 10/268,730 filed on Oct. 9, 2002, which is a continuation mals. of U.S. application Ser. No. 09/477,148 filed Jan. 4, 2000, 0009. In one embodiment of the present invention, there is now abandoned, which claims the benefit of U.S. Provisional provided a method for detecting expression of a gene in blood Application No. 60/115,125 filed on Jan. 6, 1999, now aban from a Subject, comprising the steps of: a) quantifying RNA doned. Each of these applications is incorporated herein by from a Subject blood sample; and b) detecting expression of reference in their entirety, including the figures and drawings. the gene in the quantified RNA, wherein the expression of the gene in quantified RNA indicates the expression of the gene in FIELD OF THE INVENTION the subject blood. 0010. In another embodiment of the present invention, 0002 The present invention relates generally to the there is provided a method for detecting expression of one or molecular biology of human diseases. More specifically, the more genes in blood from a Subject, comprising the steps of present invention relates to a process using the genetic infor a) obtaining a subject blood sample; b) extracting RNA from mation contained in human peripheral whole blood for the the blood sample; c) amplifying the RNA; d) generating diagnosis, prognosis and monitoring of genetic and infectious expressed sequence tags (ESTs) from the amplified RNA disease in the human body. product; and e) detecting expression of the genes in the ESTs, wherein the expression of the genes in the ESTs indicates the DESCRIPTION OF THE RELATED ART expression of the genes in the subject blood. Preferably, the 0003. The blood is a vital part of the human circulatory genes are tissue-specific genes. system for the human body. Numerous cell types make up the 0011 Instill another embodiment of the present invention, blood tissue including monocytes, leukocytes, lymphocytes there is provided a method for detecting expression of one or and erythrocytes. Although many blood cell types have been more genes in blood from a Subject, comprising the steps of a) described, there are likely many as yet undiscovered cell obtaining a subject blood sample; b) extracting DNA frag types in the human blood. Some of these undiscovered cells ments from the blood sample; c) amplifying the DNA frag may exist transiently, such as those derived from tissues and ments; and d) detecting expression of the genes in the ampli organs that are constantly interacting with the circulating fied DNA product, wherein the expression of the genes in the blood in health and disease. Thus, the blood can provide an amplified DNA product indicates the expression of the genes immediate picture of what is happening in the human body at in the subject blood. any given time. 0012. In yet another embodiment of the present invention, 0004. The turnover of cells in the hematopoietic system is there is provided a method for monitoring a course of a enormous. It was reported that over one trillion cells, includ therapeutic treatment in an individual, comprising the steps ing 200 billion erythrocytes and 70 billion neutrophilic leu of: a) obtaining a blood sample from the individual; b) kocytes, turn over each day in the human body (Ogawa 1993). extracting RNA from the blood sample; c) amplifying the As a consequence of continuous interactions between the RNA; d) generating expressed sequence tags (ESTs) from the blood and the body, genetic changes that occur within the amplified RNA product; e) detecting expression of genes in cells or tissues of the body will trigger specific changes in the ESTs, wherein the expression of the genes is associated gene expression within blood. It is the goal of the present with the effect of the therapeutic treatment; and f) repeating invention that these genetic alterations be harnessed for diag steps a)-e), wherein the course of the therapeutic treatment is nostic and prognostic purposes, which may lead to the devel monitored by detecting the change of expression of the genes opment of therapeutics for ameliorating disease. in the ESTs. Such a method may also be used for monitoring 0005. The complete profile of gene expression in the cir the onset of overt symptoms of a disease, wherein the expres culating blood remains totally unexplored. It is hypothesized sion of the genes is associated with the onset of the symptoms. that gene expression in the blood is reflective of body state 0013 Instill yet another embodiment of the present inven and, as such, the resultant disruption of homeostasis under tion, there is provided a method for diagnosing a disease in a conditions of disease can be detected through analysis of test Subject, comprising the steps of: a) generating a cDNA transcripts differentially expressed in the blood alone. Thus, library for the disease from a whole blood sample from a the identification of several key transcripts or genetic markers normal Subject; b) generating expressed sequence tag (EST) in blood will provide information about the genetic state of profile from the normal subject clNA library; c) generating a the cells, tissues, organ systems of the human body in health cDNA library for the disease from a whole blood sample from and disease. a test subject; d) generating EST profile from the test subject 0006. The prior art is deficient in non-invasive methods of cDNA library; and e) comparing the test subject EST profile screening for tissue-specific diseases. The present invention to the normal subject EST profile, wherein if the test subject fulfills this long-standing need and desire in the art. EST profile differs from the normal subject EST profile, the test Subject might be diagnosed with the disease. SUMMARY OF THE INVENTION 0014 Instill yet another embodiment of the present inven 0007. This present invention discloses a process of using tion, there is provided a kit for diagnosing, prognosing or the genetic information contained in human peripheral whole predicting a disease, comprising: a) gene-specific primers; blood in the diagnosis, prognosis and monitoring of genetic wherein the primers are designed in Such a way that their US 2010/01 24746 A1 May 20, 2010 sequences contain the opposing ends of two adjacent exons had late onset diabetes type II. FIG. 5C shows standardized for the specific gene with the intron sequence excluded; and levels of insulin gene expressed in each fractionated cell from b) a carrier, wherein the carrier immobilizes the primer(s).
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