GENE-SPECIFIC UNBIASED AMPLIFICATION METHOD

• US 20180245130A1
• Filed: 02/23/2016
• Published: 08/30/2018
• Est. Priority Date: 02/23/2015
• Status: Active Grant

• Current Assignee: Tohoku University National Universities Corporation
• Original Assignee: Tohoku University National Universities Corporation
• Inventors: Ogasawara, Koetsu
• Granted Patent: US 10,829,809 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides

Abstract

The present invention is intended to provide a method of amplifying a target gene without bias and an adapter DNA use therefor. The adapter DNA of the present invention is double-stranded adapter DNA, which is used for unbiased gene amplification and has the following features: (a) the double-stranded adapter DNA has a sense strand and an antisense strand that are annealed with each other, the base length of the sense strand being equal to or longer than the base length of the antisense strand; (b) the base length of the sense strand is 15 to 40 bp; (c) the antisense strand includes a plurality of uracil bases, each uracil is removed by treating an adapter portion with uracil DNA glycosylase (UNG), and then, heat treatment is performed to degrade the antisense strand; (d) at least one end of the adapter DNA is in the form of a blunt end; (e) the other end of the adapter DNA binds to a target gene to be amplified; and (f) a part or all of the sense strand corresponds to a forward primer sequence used for gene amplification.

Claims

1. Double-stranded adapter DNA, which is used for unbiased gene amplification and has the following features:

(a) the double-stranded adapter DNA has a sense strand and an antisense strand that are annealed with each other, the base length of the sense strand being equal to or longer than the base length of the antisense strand;

(b) the base length of the sense strand is 15 to 40 bp;

(c) the antisense strand includes a plurality of uracil bases, each uracil is removed by treating an adapter portion with uracil DNA glycosylase (UNG), and then, heat treatment is performed to degrade the antisense strand;

(d) at least one end of the adapter DNA is in the form of a blunt end;

(e) the other end of the adapter DNA binds to a target gene to be amplified; and

(f) a part or all of the sense strand corresponds to a forward primer sequence used for gene amplification.

2. The double-stranded adapter DNA according to claim 1, wherein the number of uracil bases included in the antisense strand accounts for 10% to 25% of the number of bases of the antisense strand, and uracil is present every 5 to 10 bases.

3. The double-stranded adapter DNA according to claim 1, wherein a phosphate group is bound to the 5′

end of the antisense strand and an amino group is bound to the 3′

end thereof.

4. An unbiased gene amplification kit, which includes the adapter DNA according to claim 1 and a primer comprising a part or all of a sequence of the sense strand of the adapter DNA.

6. An unbiased gene amplification kit, which includes the adapter DNA according to claim 1, a primer comprising a part or all of a sequence of the sense strand of the adapter DNA, and the an inhibitory primer having a sequence comprising:

(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′

end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified;

or

(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′

end side of the sequence of the forward primer, and

the inhibitory primer modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′

end side.

7. A method of amplifying a target gene without bias in one direction by PCR, comprising the following steps of:

(i) ligating the double-stranded adapter DNA according to claim 1 to both ends of double-stranded cDNA;

(ii) treating a gene ligated to the double-stranded adapter DNA with uracil DNA glycosylase (UNG) and further conducting heat treatment, thereby degrading the antisense strand of the adapter DNA; and

(iii) conducting PCR amplification using a forward primer comprising a part or all of a sequence of a sense strand of the double-stranded adapter DNA and a reverse primer that is specifically annealed with a target gene.

8. The method of amplifying a target gene without bias in one direction by PCR according to claim 7, wherein the reverse primer induces an elongation reaction while the forward primer alone does not induce an elongation reaction, and after a complementary strand of the sense strand of the adapter is formed, the forward primer is annealed with the complementary strand so that an elongation reaction is induced, indicating that elongation induced by the reverse primer and elongation induced by the forward primer take place in the above order in one direction.

9. The method of amplifying a target gene without bias in one direction by PCR according to claim 7, wherein the inhibitory primer has a sequence comprising:

(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′

end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified;

or

(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′

end side of the sequence of the forward primer, and

the inhibitory primer modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′

end side, is further used so that an elongation reaction induced by the forward primer alone is inhibited.

10. A method of analyzing a TCR or BCR repertoire, which comprises synthesizing cDNA from total RNA extracted from T cells or B cells, conducting comprehensive amplification of a TCR gene or BCR gene repertoire using, as a reverse primer, a primer that is specifically annealed with a C region sequence of a T cell receptor (TCR) or B cell receptor (BCR) by the method according to claim 7, and conducting sequencing using a sequencer.

5. An inhibitory primer, which is used for unbiased gene amplification by inhibiting gene amplification induced with a forward primer alone in an adapter ligation PCR amplification method in which a double-stranded adapter DNA and a forward primer comprising a partial sequence of a sense strand of the double-stranded adapter DNA are used, wherein

(a) the inhibitory primer has a sequence comprising:

(i) all or a part of a sequence of an anchor sequence portion of an anchored oligo dT primer in which an anchor sequence is ligated to the 5′

end of an oligo dT primer, the anchored oligo dT primer being used when synthesizing single-stranded cDNA from mRNA of a target gene to be amplified;

or

(ii) a partial sequence of a sense strand of a double-stranded adapter used in an adapter ligation PCR amplification method, which is a sequence present on the 3′

end side of the sequence of the forward primer, and

(b) the inhibitory primer is modified with a phosphate group, an amino group, or dideoxyl NTP on its 3′

end side.