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Patent US6498025B1 successfully refreshed from Google Patents.
US6498025B1
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Methods and compositions for cDNA synthesis
Assignee: Individual
•
Filing: 1994-04-14
•
Granted: 2002-12-24
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Expires: 2014-04-14
Abstract
Abstract Methods and compositions for synthesizing cDNA in vivo are disclosed, wherein a synthetic polynucleotide molecule which anneals in vivo to an RNA template molecule is utilized as a primer for reverse transcriptase in vivo.
Inventors
- 👤 Jeffrey E. Miller
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 05:10
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6841; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q1/6844; C12Q1/6853
Claims Analysis
Total Claims: 79
Claim 1
Independent Claim
133 words
A method for synthesizing a complementary DNA copy of an RNA template molecule which method comprises: (a) providing at least one polynucleotide molecule which functions as a primer molecule for at least one reverse transcriptase enzyme and which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; (b) providing at least one reverse transcriptase enzyme which initiates DNA synthesis in vivo using the polynucleotide molecule as a primer; (c) introducing the polynucleotide molecule into a viable target cell in the presence of the reverse transcriptase enzyme; and (d) incubating the target cell under conditions which permit the synthesis of a DNA molecule complementary to the RNA template molecule.
Claim 2
Independent Claim
133 words
A method for synthesizing a complementary DNA copy of an RNA template molecule which method comprises: (a) providing at least one polynucleotide molecule which functions as a primer molecule for at least one reverse transcriptase enzyme and which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; (b) providing at least one reverse transcriptase enzyme which initiates DNA synthesis in vivo using the polynucleotide molecule as a primer; (c) introducing the polynucleotide molecule into a viable target cell in the presence of the reverse transcriptase enzyme; and (d) incubating the target cell under conditions which permit the synthesis of a DNA molecule complementary to the RNA template molecule.
Claim 3
Dependent Claim
13 words
The method of claim 1 wherein the RNA template molecule comprises messenger RNA.
Claim 4
Dependent Claim
12 words
The method of claim 1 wherein the polynucleotide molecule comprises ribonucleic acid.
Claim 5
Dependent Claim
17 words
The method of claim 3 wherein the ribonucleic acid comprises a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 6
Dependent Claim
30 words
The method of claim 4 wherein the reverse transcriptase-cognate primer transfer RNA molecule is modified so as to anneal to the 3′ polyadenylic acid tail of a messenger RNA molecule.
Claim 7
Dependent Claim
30 words
The method of claim 5 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule comprises a primer transfer RNA molecule modified at its 3′ end with a polyribouridylic acid sequence.
Claim 8
Dependent Claim
32 words
The method of claim 6 wherein the modified primer transfer RNA molecule comprises a modified murine tRNA pro GGG molecule and wherein the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase.
Claim 9
Dependent Claim
44 words
The method of claim 6 wherein the reverse transcriptase enzyme is Moloney murine leukemia virus reverse transcriptase and the modified reverse transcriptase-cognate primer transfer RNA molecule is a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid sequence.
Claim 10
Dependent Claim
23 words
The method of claim 7 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule comprises the sequence shown in SEQ ID No.: 7.
Claim 11
Dependent Claim
18 words
The method of claim 1 wherein the polynucleotide molecule is introduced into the viable target cell by electroporation.
Claim 12
Dependent Claim
17 words
The method of claim 1 wherein the reverse transcriptase enzyme is introduced into the viable target cell.
Claim 13
Dependent Claim
17 words
The method of claim 1 wherein the reverse transcriptase enzyme is synthesized within the viable target cell.
Claim 14
Dependent Claim
22 words
The method of claim 1 wherein the reverse transcriptase enzyme and the polynucleotide molecule are introduced separately into the viable target cell.
Claim 15
Dependent Claim
22 words
The method of claim 1 wherein the reverse transcriptase enzyme and the polynucleotide molecule are concurrently introduced into the viable target cell.
Claim 16
Dependent Claim
23 words
The method of claim 14 wherein the step of introduction of the reverse transcriptase enzyme and the polynucleotide molecule is performed by electroporation.
Claim 17
Dependent Claim
13 words
The method of claim 1 wherein the viable target cells are eukaryotic cells.
Claim 18
Dependent Claim
53 words
A polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 19
Dependent Claim
53 words
A polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 20
Dependent Claim
14 words
The polynucleotide molecule of claim 17 wherein the RNA template molecule comprises messenger RNA.
Claim 21
Dependent Claim
14 words
The polynucleotide molecule of claim 17 comprising a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 22
Dependent Claim
26 words
The polynucleotide molecule of claim 17 wherein said molecule is capable of annealing in vivo to a 3′ poly (A) tail of a messenger RNA molecule.
Claim 23
Dependent Claim
25 words
The polynucleotide molecule of claim 20 wherein said molecule comprises a primer transfer RNA molecule modified at its 3′ end with a polyribouridylic acid molecule.
Claim 24
Dependent Claim
27 words
The polynucleotide molecule of claim 21 wherein said molecule comprises a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid molecule.
Claim 25
Dependent Claim
19 words
The polynucleotide molecule of claim 22 wherein said RNA molecule comprises the sequence shown in SEQ ID No: 7.
Claim 26
Dependent Claim
107 words
A kit for synthesizing in vivo a complementary DNA copy of an RNA molecule, said kit comprising: (a) a preparation of a polynucleotide molecule, which molecule anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; and (b) a preparation of at least one reverse transcriptase enzyme which initiates transcription in vivo from said polynucleotide molecule and which synthesizes a complementary DNA copy of the RNA template molecule.
Claim 27
Dependent Claim
107 words
A kit for synthesizing in vivo a complementary DNA copy of an RNA molecule, said kit comprising: (a) a preparation of a polynucleotide molecule, which molecule anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; and (b) a preparation of at least one reverse transcriptase enzyme which initiates transcription in vivo from said polynucleotide molecule and which synthesizes a complementary DNA copy of the RNA template molecule.
Claim 28
Dependent Claim
13 words
The kit of claim 24 wherein the RNA template molecule is messenger RNA.
Claim 29
Dependent Claim
17 words
The kit of claim 24 wherein said polynucleotide molecule is a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 30
Dependent Claim
45 words
The kit of claim 26 wherein said modified reverse transcriptase-cognate primer transfer RNA molecule comprises a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid molecule and wherein the reverse transcriptase enzyme is Moloney murine leukemia virus reverse transcriptase.
Claim 31
Dependent Claim
23 words
The kit of claim 27 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule comprises the sequence shown in SEQ ID No: 7.
Claim 32
Dependent Claim
36 words
The kit of claim 24 wherein the kit further comprises sufficient deoxynucleotide triphosphates to complete the synthesis of at least one complementary DNA copy of at least one endogenous RNA template molecule in a host cell.
Claim 33
Dependent Claim
35 words
The kit of claim 24 wherein the kit further comprises at least one deoxynucleotide triphosphate analog in an amount sufficient to render a complementary DNA molecule produced in a host cell capable of being detected.
Claim 34
Dependent Claim
19 words
The kit of claim 24 wherein the kit further comprises reagents for introducing the preparations into a target cell.
Claim 35
Dependent Claim
60 words
A DNA molecule comprising a first DNA sequence encoding a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 36
Dependent Claim
60 words
A DNA molecule comprising a first DNA sequence encoding a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 37
Dependent Claim
14 words
The DNA molecule of claim 32 wherein the RNA template molecule is messenger RNA.
Claim 38
Dependent Claim
18 words
The DNA molecule of claim 32 wherein the polynucleotide molecule is a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 39
Dependent Claim
27 words
The DNA molecule of claim 34 wherein the polynucleotide molecule comprises a modified primer transfer RNA molecule modified at its 3′ end with a polyribouridylic acid molecule.
Claim 40
Dependent Claim
19 words
The DNA molecule of claim 35 wherein the DNA molecule comprises the sequence shown in SEQ ID No: 8.
Claim 41
Dependent Claim
38 words
The DNA molecule of claim 32 wherein the DNA molecule further comprises a promoter sequence operatively linked to the 5′ end of the first DNA sequence, said promoter sequence capable of directing transcription of the first DNA sequence.
Claim 42
Dependent Claim
22 words
The DNA molecule of claim 37 wherein the promoter sequence is a promoter sequence at which an RNA polymerase can initiate transcription.
Claim 43
Dependent Claim
29 words
The DNA molecule of claim 37 wherein the first DNA sequence encodes a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid molecule.
Claim 44
Dependent Claim
35 words
The DNA molecule of claim 38 wherein the DNA molecule further comprises at least one restriction enzyme recognition site suitable for linearizing the DNA molecule at a point immediately 3′ of the polyribouridylic acid molecule.
Claim 45
Dependent Claim
62 words
A recombinant DNA vector which comprises a nucleotide sequence encoding a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and which functions as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 46
Dependent Claim
62 words
A recombinant DNA vector which comprises a nucleotide sequence encoding a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and which functions as a primer molecule for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 47
Dependent Claim
15 words
The recombinant DNA vector of claim 41 wherein the RNA template molecule is messenger RNA.
Claim 48
Dependent Claim
19 words
The recombinant DNA vector of claim 41 wherein the polynucleotide molecule is a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 49
Dependent Claim
29 words
The recombinant DNA vector of claim 43 wherein the polynucleotide molecule is a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid molecule.
Claim 50
Dependent Claim
67 words
A recombinant DNA vector which comprises a nucleotide sequence encoding a reverse transcriptase-cognate primer transfer RNA molecule, which molecule anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and which functions in vivo as a primer molecule for a reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 51
Dependent Claim
67 words
A recombinant DNA vector which comprises a nucleotide sequence encoding a reverse transcriptase-cognate primer transfer RNA molecule, which molecule anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and which functions in vivo as a primer molecule for a reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme.
Claim 52
Dependent Claim
26 words
The recombinant DNA vector of claim 45 wherein the reverse transcriptase-cognate primer transfer RNA molecule is modified at its 3′ end with a polyribouridylic acid molecule.
Claim 53
Dependent Claim
20 words
The recombinant DNA vector of claim 45 wherein the recombinant vector comprises the sequence shown in SEQ ID No: 8.
Claim 54
Dependent Claim
11 words
A transformed host cell containing the vector of claim 45 .
Claim 55
Dependent Claim
15 words
The transformed host cell of claim 48 wherein said host cell is a bacterial cell.
Claim 56
Dependent Claim
66 words
A synthetic polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer for an DNA polymerase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said DNA polymerase enzyme such that synthesis of a DNA molecule complementary to the RNA template molecule can occur.
Claim 57
Dependent Claim
66 words
A synthetic polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions in vivo as a primer for an DNA polymerase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said DNA polymerase enzyme such that synthesis of a DNA molecule complementary to the RNA template molecule can occur.
Claim 58
Dependent Claim
47 words
The synthetic polynucleotide molecule of claim 50 which is capable of: (a) annealing in vivo to an RNA template molecule; and (b) acting as a primer for an RNA-dependent DNA polymerase enzyme such that synthesis of a DNA molecule complementary to the RNA template molecule can occur.
Claim 59
Dependent Claim
41 words
A recombinant RNA molecule comprising: a modified tRNA molecule in which at least 3′ ribonucleoside bases are replaced with ribouridyl bases and wherein the molecule has an activity of promoting in vivo reverse transcriptase polymerase activity on an RNA template molecule.
Claim 60
Dependent Claim
41 words
A recombinant RNA molecule comprising: a modified tRNA molecule in which at least 3′ ribonucleoside bases are replaced with ribouridyl bases and wherein the molecule has an activity of promoting in vivo reverse transcriptase polymerase activity on an RNA template molecule.
Claim 61
Dependent Claim
54 words
A recombinant RNA molecule which has an activity of promoting in vivo reverse transcriptase polymerase activity on an RNA template molecule by annealing to the template molecule at a position other than a naturally-occurring retroviral primer binding site, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase polymerase enzyme.
Claim 62
Dependent Claim
54 words
A recombinant RNA molecule which has an activity of promoting in vivo reverse transcriptase polymerase activity on an RNA template molecule by annealing to the template molecule at a position other than a naturally-occurring retroviral primer binding site, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase polymerase enzyme.
Claim 63
Independent Claim
275 words
A method for producing in vivo a complementary DNA copy of an RNA template molecule of which at least a partial sequence is known comprising: (a) providing a DNA molecule comprising a gene which encodes a promoter sequence operatively linked to a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions as a primer for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; (b) providing a first nucleotide primer wherein said primer is homologous to a sequence located 5′ to the end of the promoter sequence; (c) providing a second nucleotide primer wherein said primer comprises a 3′ sequence that is complementary to a 3′ region of the polynucleotide molecule of step (a) joined to a 5′ sequence that is complementary to a portion of the known RNA template molecule sequence; (d) contacting said DNA molecule with said first and second primers; (e) treating the mixture of step (d) under conditions and with reagents which permit the amplification of said cloned DNA molecule; (f) treating at least one amplified DNA molecule produced in step (e) under conditions and with reagents which permit the production of an encoded RNA molecule; (g) introducing the RNA molecule produced in step (f) into a viable target cell in the presence of at least one reverse transcriptase enzyme which initiates DNA synthesis using said RNA molecule as a primer; and (h) incubating the target cell under conditions such that a DNA molecule complementary to at least one RNA template molecule is produced.
Claim 64
Independent Claim
275 words
A method for producing in vivo a complementary DNA copy of an RNA template molecule of which at least a partial sequence is known comprising: (a) providing a DNA molecule comprising a gene which encodes a promoter sequence operatively linked to a polynucleotide molecule which anneals in vivo to an RNA template molecule at a position other than a naturally-occurring retroviral primer binding site and functions as a primer for at least one reverse transcriptase enzyme, wherein said polynucleotide molecule comprises a stem loop structure recognized by said reverse transcriptase enzyme; (b) providing a first nucleotide primer wherein said primer is homologous to a sequence located 5′ to the end of the promoter sequence; (c) providing a second nucleotide primer wherein said primer comprises a 3′ sequence that is complementary to a 3′ region of the polynucleotide molecule of step (a) joined to a 5′ sequence that is complementary to a portion of the known RNA template molecule sequence; (d) contacting said DNA molecule with said first and second primers; (e) treating the mixture of step (d) under conditions and with reagents which permit the amplification of said cloned DNA molecule; (f) treating at least one amplified DNA molecule produced in step (e) under conditions and with reagents which permit the production of an encoded RNA molecule; (g) introducing the RNA molecule produced in step (f) into a viable target cell in the presence of at least one reverse transcriptase enzyme which initiates DNA synthesis using said RNA molecule as a primer; and (h) incubating the target cell under conditions such that a DNA molecule complementary to at least one RNA template molecule is produced.
Claim 65
Dependent Claim
13 words
The method of claim 54 wherein the RNA template molecule comprises messenger RNA.
Claim 66
Dependent Claim
12 words
The method of claim 54 wherein the polynucleotide molecule comprises ribonucleic acid.
Claim 67
Dependent Claim
17 words
The method of claim 56 wherein the ribonucleic acid comprises a reverse transcriptase-cognate primer transfer RNA molecule.
Claim 68
Dependent Claim
28 words
The method of claim 57 wherein the reverse transcriptase-cognate primer transfer RNA molecule is capable of annealing to the 3′ polyadenylic acid tail of a messenger RNA molecule.
Claim 69
Dependent Claim
30 words
The method of claim 58 wherein the reverse transcriptase-cognate primer transfer RNA molecule comprises a modified primer transfer RNA molecule modified at its 3′ end with a polyribouridylic acid sequence.
Claim 70
Dependent Claim
32 words
The method of claim 59 wherein the modified primer transfer RNA molecule comprises a modified murine tRNA pro GGG molecule and wherein the reverse transcriptase is Moloney murine leukemia virus reverse transcriptase.
Claim 71
Dependent Claim
44 words
The method of claim 60 wherein the reverse transcriptase enzyme is Moloney murine leukemia virus reverse transcriptase and the modified reverse transcriptase-cognate primer transfer RNA molecule is a modified murine tRNA pro GGG molecule modified at its 3′ end with a polyribouridylic acid sequence.
Claim 72
Dependent Claim
23 words
The method of claim 61 wherein the modified reverse transcriptase-cognate primer transfer RNA molecule comprises the sequence shown in SEQ ID No.: 7.
Claim 73
Dependent Claim
21 words
The method of claim 54 wherein the RNA molecule of step (g) is introduced into the viable target cell by electroporation.
Claim 74
Dependent Claim
17 words
The method of claim 54 wherein the reverse transcriptase enzyme is introduced into the viable target cell.
Claim 75
Dependent Claim
17 words
The method of claim 54 wherein the reverse transcriptase enzyme is synthesized within the viable target cell.
Claim 76
Dependent Claim
22 words
The method of claim 54 wherein the reverse transcriptase enzyme and the polynucleotide molecule are introduced separately into the viable target cell.
Claim 77
Dependent Claim
22 words
The method of claim 54 wherein the reverse transcriptase enzyme and the polynucleotide molecule are concurrently introduced into the viable target cell.
Claim 78
Dependent Claim
23 words
The method of claim 67 wherein the step of introduction of the reverse transcriptase enzyme and the polynucleotide molecule is performed by electroporation.
Claim 79
Dependent Claim
13 words
The method of claim 54 wherein the viable target cells are eukaryotic cells.
References Cited (Backward Citations)
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