Appeal point
A method capable of sequencing and quantifying a target nucleic acid without carrying out a ligation reaction.
Summary
It is an object of the present invention to provide a method that combines sequencing of a target nucleic acid and quantification of the target nucleic acid without performing a ligation reaction and solves a problem caused by PCR amplification efficiency. The present invention is a method for quantifying a target nucleic acid, which includes the following steps 1 to 3.
1) A single primer extension reaction is carried out using a primer having a sequence arrangement specific to the sequence of the target nucleic acid and having a random sequence and a known sequence introduced upstream thereof. As a result, a double-stranded nucleic acid having a random sequence and the sequence of the target nucleic acid is obtained.
2) The obtained double-stranded nucleic acid is used as a template to carry out PCR using a primer specific to a known sequence and a primer having a part of the sequence of the target nucleic acid. An amplified product of double-stranded nucleic acid having a random sequence and a sequence of the target nucleic acid is obtained.
3) The random sequence together with the sequence of the target nucleic acid are decoded by sequencing using the obtained amplified product of the double-stranded nucleic acid.
1) A single primer extension reaction is carried out using a primer having a sequence arrangement specific to the sequence of the target nucleic acid and having a random sequence and a known sequence introduced upstream thereof. As a result, a double-stranded nucleic acid having a random sequence and the sequence of the target nucleic acid is obtained.
2) The obtained double-stranded nucleic acid is used as a template to carry out PCR using a primer specific to a known sequence and a primer having a part of the sequence of the target nucleic acid. An amplified product of double-stranded nucleic acid having a random sequence and a sequence of the target nucleic acid is obtained.
3) The random sequence together with the sequence of the target nucleic acid are decoded by sequencing using the obtained amplified product of the double-stranded nucleic acid.
Schematic image
Application
Ecology, biology, medical
Relational patent
Item name
:
Method for quantifying target nucleic acid and kit therefor
Filing date
:
2015-11-18
Inventor(s)
Tatsuhiko Hoshino
Fumio Inagaki