Method for fragmenting double-stranded RNA and use of the same
AssigneeJapan Agency for Marine-Earth Science and Technology
InventorsShunichi Urayama, Takuro Nunoura, Shigeru Deguchi
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Abstract
The object is to provide a method that enables detection of unknown virus sequences and efficient detection and search of viruses. The method comprises the step of randomly fragmenting an objective double-stranded (ds) RNA to obtain dsRNA fragments; the step of subjecting the obtained dsRNA fragments to a reverse transcription reaction and then performing polymerase chain reaction (PCR) to obtain corresponding DNA fragments; and the step of subjecting the obtained DNA fragments to a sequence analysis operation to determine the sequences. The reverse transcription reaction is preferably started from the 3′ ends of the dsRNA fragments.
Claims13
A method for determining an RNA sequence, which comprises:
- separating double-stranded RNA (dsRNA) in a sample from DNA and single-stranded RNA to obtain purified dsRNA;
- randomly fragmenting the purified dsRNA to obtain dsRNA fragments, wherein the obtained dsRNA fragments have a 150- to 5000-base length;
- subjecting the obtained dsRNA fragments to a reverse transcription reaction starting from the 3′ ends of the dsRNA fragments, and then performing polymerase chain reaction (PCR) to obtain corresponding DNA fragments;
- subjecting the obtained DNA fragments to a sequence analysis operation to determine sequences of the fragments;
- and
- comparing the determined sequences with a known virus sequence.
A method for preparing DNA fragments, which comprises:
- separating a double-stranded RNA (dsRNA) in a sample from DNA and single-stranded RNA to obtain purified dsRNA wherein the obtained dsRNA fragments have a 150-5000 base length;
- randomly fragmenting the purified dsRNA to obtain dsRNA fragments;
- and
- subjecting the obtained dsRNA fragments to a reverse transcription reaction starting from the 3′ ends of the dsRNA fragments and then performing polymerase chain reaction (PCR) to obtain corresponding DNA fragments.
A method for analyzing a virus, which comprises:
- separating dsRNA in a sample from DNA and single-stranded RNA to obtain purified dsRNA;
- randomly fragmenting the purified dsRNA to obtain dsRNA fragments, wherein the obtained dsRNA fragments have a 150- to 5000-based length;
- subjecting the obtained dsRNA fragments to a reverse transcription reaction starting from the 3′ ends of the dsRNA fragments, and then performing polymerase chain reaction (PCR) to obtain corresponding DNA fragments;
- subjecting the obtained DNA fragments to a sequence analysis operation to determine sequences of the fragments;
- and
- analyzing presence or absence and/or characteristic of a virus in the sample on the basis of the determined sequences.
Dependencies are inferred from the claim text.