Promoter ligation activated transcription amplification of nucleic acid sequences
AssigneeLife Technologies Corporation
InventorsMark S. Berninger, David M. Schuster, Ayoub Rashtchian
Life of this patent
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Abstract
This invention discloses a scheme for producing nucleic acid end products that are functionally or exactly identical to the starting products, thereby resulting in exponential amplification of a desired nucleic acid sequence. Specifically, sequences are cycled between RNA and DNA forms using the following basic steps: (1) a T7 RNA polymerase promoter is ligated onto a single-stranded DNA template; (2) T7 RNA polymerase makes many copies of RNA: (3) a complementary DNA is made from the RNA by extension of a primer by reverse transcriptase; and (4) the RNA template is removed by ribonuclease H. This amplification method is useful for purposes such as genetic research and diagnostic assays.
Claims19
A method for amplifying and detecting a target DNA sequence present, or possibly present, in a nucleic acid sample in a reaction volume under incubation conditions, comprising:
- (a) adding under hybridizing conditions to said sample the following components:
- (1) a primer,
- (2) a proto-promoter comprising
- (a) a double-stranded segment which encodes a promoter for a DNA dependent RNA polymerase, and
- (b) a single-stranded segment which is complementary to the 3' end of the target DNA and which is a 3' overhand;
- (3) ribonucleotide triphosphates,
- (4) deoxyribonucleotide triphosphates,
- (5) RNA-dependent DNA polymerase,
- (6) an enzyme capable of releasing single-stranded DNA from RNA-DNA heteroduplexes,
- (7) DNA ligase,
- (8) DNA-dependent RNA polymerase, and
- (9) buffer solution;
- (b) annealing together said proto-promoter and target DNA, thereby forming a target DNA/proto-promoter complex;
- (c) ligating together the target DNA/photo-promoter complex, thereby forming a covalently-bound target DNA/proto-promoter combination;
- (d) transcribing the covalently-bound target DNA/proto-promoter combination with DNA-dependent RNA polymerase, thereby forming transcribed RNA;
- (e) annealing the primer to the transcribed RNA, thereby forming a reverse transcription initiation complex;
- (f) reverse transcribing the RNA with RNA-dependent DNA polymerase by extension of the primer of the reverse transcription initiation complex to form additional DNA, thereby forming a DNA-RNA heteroduplex;
- (g) releasing the DNA strand of the DNA-RNA heteroduplex, thereby liberating the additional DNA;
- (h) repeating step (b) through (g), thereby providing a cyclical process;
- and
- (i) detecting nucleic acids synthesized from said cyclical process steps (b) through (h);
- wherein said steps (b) through (h) are carried out without temperature cycling.
A method for amplifying and detecting a target RNA sequence present, or possibly present, in a nucleic acid sample in a reaction volume under incubation conditions, comprising:
- (a) adding under hybridizing conditions to said sample the following components:
- (1) a primer,
- (2) a proto-promoter comprising
- (a) a double-stranded segment which encodes a promoter for a DNA dependent RNA polymerase, and
- (b) a single-stranded segment which is complementary to the 3' end of copy DNA reverse transcribed from target RNA and which is a 3' overhang;
- (3) ribonucleotide triphosphates,
- (4) deoxyribonucleotide triphosphates,
- (5) RNA-dependent DNA polymerase,
- (6) an enzyme capable of releasing single-stranded DNA from RNA-DNA heteroduplexes,
- (7) DNA ligase,
- (8) DNA-dependent RNA polymerase, and
- (9) buffer solution;
- (b) annealing the primer to target RNA, thereby forming a first transcription initiation complex;
- (c) reverse transcribing target RNA with RNA-dependent DNA polymerase by extension of the primer of the reverse transcription initiation complex to form copy DNA, thereby forming a DNA-RNA heteroduplex;
- (d) releasing the DNA strand of the DNA-RNA heteroduplex, thereby liberating copy DNA;
- (e) annealing together said proto-promoter and the copy DNA, thereby forming a copy DNA/proto-promoter complex;
- (f) ligating together the copy DNA/proto-promoter complex, thereby forming a covalently bound copy DNA/proto-promoter combination;
- (g) transcribing the covalently bound copy DNA/proto-promoter combination with DNA-dependent RNA polymerase, thereby forming additional RNA;
- (h) repeating steps (b) through (g), thereby providing a cyclical process;
- and
- (i) detecting nucleic acids synthesized from said cyclical process steps (b) through (h);
- wherein said steps (b) through (h) are carried out without temperature cycling.
Dependencies are inferred from the claim text.