US 2025/0051850 A1Published application

Method for analyzing structural variation, primer pair set, and method for designing primer pair set

AssigneeThe University of Tokyo

InventorKazuaki YOKOYAMA

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Abstract

An analysis method suitable for each SV is provided by utilizing a structural characteristic of the SV. The analysis method is a method for analyzing the presence/absence of a specific structural variation in a sample DNA including a genomic DNA, a free DNA, or a complementary DNA derived from a subject, and including a detection step of detecting the presence/absence of the specific structural variation by performing PCR using the sample DNA as a template, and using, as a primer, a primer pair set consisting of a first primer pair and a second primer pair.

Claims17

Dependent claims

A method for analyzing the presence/absence of a specific structural variation in a sample DNA including a genomic DNA, a free DNA or a complementary DNA derived from a subject, comprising:

a detection step of detecting the presence/absence of the specific structural variation by performing PCR using, as a template, the sample DNA, and using, as a primer, a primer pair set consisting of a first primer pair and a second primer pair,
wherein the specific structural variation includes at least one selected from the group consisting of insertion, translocation, deletion, inversion, and tandem duplication, and the primer pair set is selected in accordance with the specific structural variation,
the first primer pair consists of a forward primer (WT-F) and a reverse primer (WT-R) that anneal at positions for amplifying a target sequence in a wild-type nucleotide sequence not including the specific structural variation,
the second primer pair consists of a forward primer (Mut-F) and a reverse primer (Mut-R) that anneal at positions for amplifying a target sequence in a mutant nucleotide sequence including the specific structural variation, and
the primer pair set includes one or more selected from the group consisting of:
a primer pair set (A) consisting of the first primer pair designed so that a length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is 10 times or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (B) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (C) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the length of the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) when the specific structural variation is included is 1/10 or less than that when the specific structural variation is not included;
and
a primer pair set (D) consisting of the first primer pair designed so that the length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is twice or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included.

7 more elements

A primer pair set for use in PCR for analyzing the presence/absence of a specific structural variation in a sample DNA including a genomic DNA, a free DNA, or a complementary DNA derived from a subject,

the specific structural variation including at least one selected from the group consisting of insertion, translocation, deletion, inversion, and tandem duplication,
the primer pair set comprising a first primer pair and a second primer pair,
wherein the first primer pair consists of a forward primer (WT-F) and a reverse primer (WT-R) that anneal at positions for amplifying a target sequence in a wild-type nucleotide sequence not including the specific structural variation,
the second primer pair consists of a forward primer (Mut-F) and a reverse primer (Mut-R) that anneal at positions for amplifying a target sequence in a mutant nucleotide sequence including the specific structural variation, and
the primer pair set is one or more selected from the group consisting of:
a primer pair set (A) consisting of the first primer pair designed so that a length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is 10 times or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (B) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (C) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the length of the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) when the specific structural variation is included is 1/10 or less than that when the specific structural variation is not included;
and
a primer pair set (D) consisting of the first primer pair designed so that the length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is twice or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included.

7 more elements

A method for designing a primer pair set for use in PCR for analyzing the presence/absence of a specific structural variation in a sample DNA including a genomic DNA, a free DNA, or a complementary DNA derived from a subject,

wherein the specific structural variation includes at least one selected from the group consisting of insertion, translocation, deletion, inversion, and tandem duplication,
the primer pair set comprises a first primer pair and a second primer pair,
the first primer pair consists of a forward primer (WT-F) and a reverse primer (WT-R) that anneal at positions for amplifying a target sequence in a wild-type nucleotide sequence not including the specific structural variation,
the second primer pair consists of a forward primer (Mut-F) and a reverse primer (Mut-R) that anneal at positions for amplifying a target sequence in a mutant nucleotide sequence including the specific structural variation, and
the primer pair set is one or more selected from the group consisting of:
a primer pair set (A) consisting of the first primer pair designed so that a length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is 10 times or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (B) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (C) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the length of the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) when the specific structural variation is included is 1/10 or less than that when the specific structural variation is not included;
and
a primer pair set (D) consisting of the first primer pair designed so that the length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is twice or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included.

7 more elements

Dependencies are inferred from the claim text.

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Abstract

A method for analyzing the presence/absence of a specific structural variation in a sample DNA including a genomic DNA, a free DNA or a complementary DNA derived from a subject, comprising:

a detection step of detecting the presence/absence of the specific structural variation by performing PCR using, as a template, the sample DNA, and using, as a primer, a primer pair set consisting of a first primer pair and a second primer pair,
wherein the specific structural variation includes at least one selected from the group consisting of insertion, translocation, deletion, inversion, and tandem duplication, and the primer pair set is selected in accordance with the specific structural variation,
the first primer pair consists of a forward primer (WT-F) and a reverse primer (WT-R) that anneal at positions for amplifying a target sequence in a wild-type nucleotide sequence not including the specific structural variation,
the second primer pair consists of a forward primer (Mut-F) and a reverse primer (Mut-R) that anneal at positions for amplifying a target sequence in a mutant nucleotide sequence including the specific structural variation, and
the primer pair set includes one or more selected from the group consisting of:
a primer pair set (A) consisting of the first primer pair designed so that a length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is 10 times or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (B) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included;
a primer pair set (C) consisting of the first primer pair designed so that the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) is present only when the specific structural variation is not included, and the second primer pair designed so that the length of the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) when the specific structural variation is included is 1/10 or less than that when the specific structural variation is not included;
and
a primer pair set (D) consisting of the first primer pair designed so that the length of the target sequence to be amplified by the forward primer (WT-F) and the reverse primer (WT-R) when the specific structural variation is included is twice or more than that when the specific structural variation is not included, and the second primer pair designed so that the target sequence to be amplified by the forward primer (Mut-F) and the reverse primer (Mut-R) is present only when the specific structural variation is included.

7 more elements