Ligation-Based Rna Amplification
First Claim
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1. A method of producing a ligated nucleic acid molecule comprising:
- a) supplying RNA other than poly A;
b) supplying one or more nucleic acids having a double stranded region and a single stranded 3′
terminal region; and
c) hybridizing the single stranded 3′
terminal region of the nucleic acid sequence to the RNA and ligating one 5′
end of the double stranded region of the nucleic acid to the 3′
end of the RNA by enzymatic means.
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Abstract
Methods of amplification, purification and detection of nucleic acid sequences especially RNA are described. One aspect of the method involves the hybridisation and subsequent ligation of a nucleic acid structure to the nucleic acid sequence desired to be manipulated. The methods require that the nucleic acid structure comprises a double stranded region and a single stranded region. The single stranded region is complementary to the RNA sequence of interest. The double stranded region may also contain additional functionalities which are then used subsequently in the method.
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Citations
33 Claims
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1. A method of producing a ligated nucleic acid molecule comprising:
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a) supplying RNA other than poly A;
b) supplying one or more nucleic acids having a double stranded region and a single stranded 3′
terminal region; and
c) hybridizing the single stranded 3′
terminal region of the nucleic acid sequence to the RNA and ligating one 5′
end of the double stranded region of the nucleic acid to the 3′
end of the RNA by enzymatic means. - View Dependent Claims (2, 3, 4, 5, 6, 7, 25)
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8. A method of amplifying a target RNA sequence comprising the steps of:
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a) supplying the RNA in single stranded form;
b) adding a DNA sequence that comprises a double stranded region which contains a promoter sequence for RNA polymerase and a single stranded region which hybridizes to the target RNA;
c) ligating the DNA sequence to the 3′
end of RNA by enzymatic means to produce a DNA-RNA; and
d) transcribing the DNA-RNA with RNA polymerase to produce antisense complementary RNA (cRNA). - View Dependent Claims (9, 10, 11, 12, 13, 14, 22)
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15-21. -21. (canceled)
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23-24. -24. (canceled)
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26. A method of analyzing nucleic acid comprising:
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a) hybridizing an oligodeoxyribonucleotide which contains natural and modified nucleotides to an RNA sequence such that the oligodeoxyribonucleotide is complementary to a portion of the RNA;
b) contacting the resulting RNA-DNA heteroduplex with an agent that specifically nicks only the RNA strand; and
c) ligating a DNA sequence to the trimmed RNA 3′
end of step b). - View Dependent Claims (27)
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28. A method of analyzing nucleic acid comprising:
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a) hybridizing an oligodeoxyribonucleotide which contains natural and modified nucleotides to an RNA sequence such that the oligodeoxyribonucleotide is complementary to a portion of the RNA and that the oligodeoxyribonucleotide hybridises at the poly(A) tail;
message junctionb) contacting the resulting RNA-DNA heteroduplex with an agent that specifically nicks only the RNA strand at the poly(A);
message junctionc) determining the size of the poly(A) tail.
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- 29. A kit comprising a nucleic acid sequence that comprises or nucleic acid sequences that comprise a double stranded region and a single stranded region and a DNA ligase.
Specification