US6221600B1
Expired / Lapsed
US
⏱️ 0.0 years remaining
Combinatorial oligonucleotide PCR: a method for rapid, global expression analysis
Assignee: The University of Texas System
•
Filing: 1999-10-08
•
Granted: 2001-04-24
•
Expires: 2019-10-08
Abstract
Abstract The present invention relates to a method for the detection of gene expression and analysis of both known and unknown genes. The invention is a highly sensitive, rapid and cost-effective means of monitoring gene expression, as well as for the analysis and quantitation of changes in gene expression for a defined set of genes and in response to a wide variety of events. It is an important feature of the present invention that no single molecular species of cDNA gives rise to more than one fragment in the collection of products which are subsequently amplified and representative of each expressed gene. This achievement is facilitated by immobilizing the cDNA prior to digesting and then digesting with sequentially with two frequently cutting enzymes. Linker oligomers are ligated to each cut site following the respective digestion. Primers, complementary to the oligomer sequence with an additional 3' variable sequence are used to amplify the fragments. Using an array of fragments theoretically facilitates the amplification of all of the possible messages in a given sample.
Inventors
- 👤 Michael C. MacLeod
- 👤 C. Marcelo Aldaz
- 👤 Sara S. Gaddis
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 10:28
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6809
Claims Analysis
Total Claims: 86
Claim 1
Independent Claim
72 words
A method comprising: a) obtaining a DNA comprising an anchorable moiety; b) cleaving said DNA with a first restriction endonuclease; c) ligating a linker molecule to cleaved DNA produced in step b; d) immobilizing linker ligated DNA through said anchorable moiety; e) cleaving DNA immobilized in step d with a second restriction endonuclease; f) ligating a second linker molecule to DNA cleaved in step e; g) amplfying DNA ligated in step f.
Claim 2
Independent Claim
72 words
A method comprising: a) obtaining a DNA comprising an anchorable moiety; b) cleaving said DNA with a first restriction endonuclease; c) ligating a linker molecule to cleaved DNA produced in step b; d) immobilizing linker ligated DNA through said anchorable moiety; e) cleaving DNA immobilized in step d with a second restriction endonuclease; f) ligating a second linker molecule to DNA cleaved in step e; g) amplfying DNA ligated in step f.
Claim 3
Dependent Claim
18 words
The method of claim 1 , wherein said DNA is immobilized prior to cleaving with said first endonuclease.
Claim 4
Dependent Claim
12 words
The method of claim 1 , wherein said DNA is non-genomic DNA.
Claim 5
Dependent Claim
11 words
The method of claim 1 , wherein said DNA is cDNA.
Claim 6
Dependent Claim
15 words
The method of claim 1 , wherein said anchorable moiety comprises a means of adhering.
Claim 7
Dependent Claim
19 words
The method of claim 5 , wherein said means of adhering comprises a means of establishing a non-covalent interaction.
Claim 8
Dependent Claim
19 words
The method of claim 5 , wherein said means of adhering comprises a means of establishing a covalent interaction.
Claim 9
Dependent Claim
14 words
The method of claim 5 , wherein said means of adhering comprises a ligand.
Claim 10
Dependent Claim
13 words
The method of claim 5 , wherein said means of adhering is biotin.
Claim 11
Dependent Claim
14 words
The method of claim 5 , wherein said means of adhering comprises an antibody.
Claim 12
Dependent Claim
16 words
The method of claim 1 , wherein said anchorable moiety is located at the 3′ end.
Claim 13
Dependent Claim
15 words
The method of claim 4 , wherein said cDNA is reverse transcribed from messenger RNA.
Claim 14
Dependent Claim
16 words
The method of claim 12 , wherein said reverse transcription is initiated at an oligo dT.
Claim 15
Dependent Claim
16 words
The method of claim 12 , wherein said reverse transcription is initiated at a random hexamer.
Claim 16
Dependent Claim
12 words
The method of claim 13 , wherein said oligo dT is biotinylated.
Claim 17
Dependent Claim
20 words
The method of claim 15 wherein said cDNA is immobilized on a substrate by means of said biotinylated oligo dT.
Claim 18
Dependent Claim
11 words
The method of claim 16 , wherein said substrate is streptavidin.
Claim 19
Dependent Claim
19 words
The method of claim 1 , wherein the order of said first and said second restriction endonucleases is reversed.
Claim 20
Dependent Claim
25 words
The method of claim 1 , wherein said amplification is initiated at primers comprising a sequence complementary to said first and said second linkers respectively.
Claim 21
Dependent Claim
70 words
The method of claim 1 , wherein amplification is carried out with a primer set comprising: a) a first amplification primer, wherein the 5′ sequence of said primer is complementary to said first linker sequence and the 3′ sequence comprises a specificity region; b) a second amplification primer, wherein the 5′ sequence of said primer is complementary to said second linker sequence and the 3′ sequence comprises a specificity region.
Claim 22
Dependent Claim
20 words
The method of claim 20 , wherein said amplification is performed with an array of combinations of alternate amplification primers.
Claim 23
Dependent Claim
11 words
The method of claim 20 wherein said DNA fragment is preamplified.
Claim 24
Dependent Claim
12 words
The method of claim 1 , further comprising identifying the amplified DNA.
Claim 25
Dependent Claim
13 words
The method of claim 23 , wherein said identification is based upon length.
Claim 26
Dependent Claim
15 words
The method of claim 23 , wherein said identification is performed by a computer program.
Claim 27
Dependent Claim
15 words
The method of claim 21 , wherein said amplification is performed in a multi-well plate.
Claim 28
Dependent Claim
22 words
The method of claim 20 , wherein the specificity region of the first amplification primer is 3,4,5,6,7 or 8 base pairs long.
Claim 29
Dependent Claim
22 words
The method of claim 20 , wherein the specificity region of the second amplification primer is 3,4,5,6,7 or 8 base pairs long.
Claim 30
Dependent Claim
28 words
The method of claim 1 , wherein said amplification comprises polymerase chain reaction, nucleic acid sequence based amplification, transcription mediated amplification, strand displacement amplification or ligase chain reaction.
Claim 31
Dependent Claim
18 words
The method of claim 1 , wherein said first restriction endonuclease has a four base pair recognition site.
Claim 32
Dependent Claim
23 words
The method of claim 1 , wherein said first restriction endonuclease has a recognition site of five, six, seven or eight base pairs.
Claim 33
Dependent Claim
29 words
The method of claim 30 , wherein said first restriction endonuclease is NlaIII, DpnII, Sau3AI Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.
Claim 34
Dependent Claim
18 words
The method of claim 1 , wherein said second restriction endonuclease has a four base pair recognition site.
Claim 35
Dependent Claim
23 words
The method of claim 1 , wherein said second restriction endonuclease has a recognition site of five, six, seven or eight base pairs.
Claim 36
Dependent Claim
28 words
The method of claim 33 , wherein the restriction endonuclease is NlaIII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.
Claim 37
Dependent Claim
15 words
The method of claim 1 , wherein a label is incorporated into said amplified DNA.
Claim 38
Dependent Claim
17 words
The method of claim 36 , wherein said label is incorporated by means of a labeled primer.
Claim 39
Dependent Claim
22 words
The method of claim 36 , further comprising partial nucleotide sequence identification of the amplified products by the identity of the label.
Claim 40
Dependent Claim
12 words
The method of claim 36 , wherein said label is a chromophore.
Claim 41
Dependent Claim
12 words
The method of claim 36 , wherein said label is a fluorophore.
Claim 42
Dependent Claim
13 words
The method of claim 36 , wherein said label is an affinity label.
Claim 43
Dependent Claim
12 words
The method of claim 36 , wherein said label is a dye.
Claim 44
Dependent Claim
38 words
The method of claim 37 , wherein the 5′ end of said primer comprises an amino moiety and a flurophore is covalently attached by the reaction of a succinimido ester of the flurophore to the 5′ amino-modified primer.
Claim 45
Dependent Claim
54 words
The method of claim 40 , wherein said fluorophore is Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, or Texas Red.
Claim 46
Dependent Claim
14 words
The method of claim 1 , wherein the products of said amplification are analyzed.
Claim 47
Dependent Claim
17 words
The method of claim 45 , wherein said analysis of amplification products is by polyacrylamide gel electrophoresis.
Claim 48
Dependent Claim
17 words
The method of claim 45 , wherein said analysis of amplification products is by capillary gel electrophoresis.
Claim 49
Dependent Claim
16 words
The method of claim 45 , wherein said analysis of amplification products is by mass spectrophotometry.
Claim 50
Dependent Claim
16 words
The method of claim 45 , wherein said analysis of amplification products is by energy transfer.
Claim 51
Independent Claim
16 words
The method of claim, 45 , wherein said analysis of amplification products is by OIA technology.
Claim 52
Dependent Claim
16 words
The method of claim 45 , wherein said analysis of amplification products utilizes fluorescently-labeled latex beads.
Claim 53
Dependent Claim
16 words
The method of claim 45 , wherein said analysis of amplification products comprises quantifying amplification products.
Claim 54
Dependent Claim
22 words
The method of claim 52 , wherein said quantifying is by measuring the ratio of each amplified product to a co-amplified reference-gene.
Claim 55
Dependent Claim
24 words
The method of claim 52 , wherein said quantifying is by measuring the ratio of each amplified product to a panel of co-amplified reference-genes.
Claim 56
Dependent Claim
16 words
The method of claim 52 , wherein said analysis of amplification products is by Real-Time PCR.
Claim 57
Dependent Claim
18 words
The method of claim 45 , wherein said analysis of amplification products is performed in a multi-well plate.
Claim 58
Dependent Claim
17 words
The method of claim 45 , wherein said analysis of amplification products is performed on a membrane.
Claim 59
Dependent Claim
18 words
The method of claim 45 , wherein said analysis of amplification products is performed on a solid matrice.
Claim 60
Dependent Claim
14 words
The method of claim 58 , wherein said solid matrice is a DNA chip.
Claim 61
Dependent Claim
26 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a different cell or tissue.
Claim 62
Dependent Claim
26 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cancerous cell or tissue.
Claim 63
Dependent Claim
29 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived a cell or tissue treated with a pharmaceutical compound.
Claim 64
Dependent Claim
30 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a teratogenic compound.
Claim 65
Dependent Claim
30 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a carcinogenic compound.
Claim 66
Dependent Claim
30 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a toxic compound.
Claim 67
Dependent Claim
31 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a biological response modifier.
Claim 68
Dependent Claim
36 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a hormone, a hormone agonist or a hormone antagonist.
Claim 69
Dependent Claim
29 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a cytokine.
Claim 70
Dependent Claim
30 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a growth factor.
Claim 71
Dependent Claim
35 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on the DNA derived from a cell or tissue treated with the ligand of a known biological receptor.
Claim 72
Dependent Claim
21 words
The method of claim 1 , performed on DNA derived from a cell or tissue type obtained from a different species.
Claim 73
Dependent Claim
21 words
The method of claim 1 , performed on DNA derived from a cell or tissue type obtained from a different organism.
Claim 74
Dependent Claim
20 words
The method of claim 1 , performed on DNA derived from a cell or tissue at different stages of development.
Claim 75
Dependent Claim
29 words
The method of claim 1 , performed on DNA derived from a normal cell or tissue and on the DNA derived from a cell or tissue that is diseased.
Claim 76
Dependent Claim
21 words
The method of claim 1 , performed on DNA derived from a cell or tissue cultured in vitro under different conditions.
Claim 77
Dependent Claim
28 words
The method of claim 1 , performed on the DNA derived from a cell or tissue from two organisms of the same species with a known genetic difference.
Claim 78
Dependent Claim
94 words
A kit for detection of gene expression comprising: a) a first restriction enzyme; b) a second restriction enzyme; c) a first, ligatable, oligonucleotide tag; d) a second, ligatable, oligonucleotide tag; e) a first amplification primer, wherein the 5′ sequence of said primer is complementary to said first oligonucleotide tag and the 3′ sequence comprises a specificity region; f) a second amplification primer, wherein the 5′ sequence of said primer is complementary to said second oligonucleotide tar and the 3′ sequence comprises a specificity region; g) software capable of analyzing data generated from said kit.
Claim 79
Dependent Claim
94 words
A kit for detection of gene expression comprising: a) a first restriction enzyme; b) a second restriction enzyme; c) a first, ligatable, oligonucleotide tag; d) a second, ligatable, oligonucleotide tag; e) a first amplification primer, wherein the 5′ sequence of said primer is complementary to said first oligonucleotide tag and the 3′ sequence comprises a specificity region; f) a second amplification primer, wherein the 5′ sequence of said primer is complementary to said second oligonucleotide tar and the 3′ sequence comprises a specificity region; g) software capable of analyzing data generated from said kit.
Claim 80
Dependent Claim
17 words
The kit of claim 77 , wherein said first restriction enzyme is a four base pair cutter.
Claim 81
Dependent Claim
29 words
The kit of claim 78 , wherein said first restriction endonuclease is NlaIII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.
Claim 82
Dependent Claim
17 words
The kit of claim 77 , wherein said second restriction enzyme is a four base pair cutter.
Claim 83
Dependent Claim
29 words
The kit of claim 80 , wherein said second restriction endonuclease is NlaII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.
Claim 84
Dependent Claim
19 words
The kit of claim 77 , wherein said first amplification primer comprises the sequence GCTGTCTAGACG (SEQ ID NO: 1).
Claim 85
Dependent Claim
18 words
The kit of claim 77 , wherein said second amplification primer comprises the sequence CGGTGATGCATC (SEQ ID NO:2).
Claim 86
Dependent Claim
16 words
The method of claim 1 , wherein said anchorable moiety is located at the 5′ end.
References Cited (Backward Citations)
US5053336A
Cited
US5290677A
Cited
EP0534858A1
Cited
US5521084A
Cited
US5804380A
Cited
US5688648A
Cited
US5656470A
Cited
US5837836A
Cited
US5874215A
Cited
US5817462A
Cited
US5843773A
Cited
US5866330A
Cited
US5736330A
Cited
US5871697A
Cited
US6007996A
Cited
US5767288A
Cited
US5851772A
Cited
WO1998008981A1
Cited
US5853992A
Cited
US5770716A
Cited
US6002817A
Cited
US5945290A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2004-04-23 | 2004-10-23 | 2005-04-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-04-23 | 2008-10-23 | 2009-04-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-04-23 | 2012-10-23 | 2013-04-24 | $7700.00 | Window Closed / Lapsed |