阅读说明：本技术 用于检测低频突变的靶向富集方法和试剂盒 (Targeted enrichment method and kit for detecting low-frequency mutation ) 是由 杨林 高雅 蒲丹丹 张海萍 程云阳 陈芳 蒋慧 于 2017-07-21 设计创作，主要内容包括：本发明提供了一种用于检测低频突变的靶向富集方法和试剂盒。该方法包括：在末端转移酶的作用下,在单链DNA和/或双链DNA每条链的3’端加上一段单一碱基的单聚核苷酸尾巴；以上游特异性引物和第一通用引物对突变目标位点所在的区域进行第一次PCR扩增,其中第一通用引物包括5’端的测序引物序列1和3’端的与单聚核苷酸尾巴互补的连续单一碱基；以下游特异性引物和第二通用引物对第一次PCR扩增的产物进行第二次PCR扩增,其中第二通用引物包括第一通用引物的测序引物序列1。(The invention provides a targeted enrichment method and a kit for detecting low-frequency mutation. The method comprises the following steps: under the action of terminal transferase, adding a single-nucleotide tail with single base at the 3' end of each strand of the single-stranded DNA and/or the double-stranded DNA; performing first PCR amplification on a region where a mutation target site is located by using an upstream specific primer and a first universal primer, wherein the first universal primer comprises a sequencing primer sequence 1 at a 5 'end and continuous single bases which are complementary with a single nucleotide tail at a 3' end; performing a second PCR amplification on the product of the first PCR amplification with a downstream specific primer and a second universal primer, wherein the second universal primer comprises the sequencing primer sequence 1 of the first universal primer.)

A targeted enrichment method for detecting low frequency mutations, comprising:

under the action of terminal transferase, adding a single-nucleotide tail with single base at the 3' end of each strand of the single-stranded DNA and/or the double-stranded DNA;

performing a first PCR amplification on a region where a mutation target site is located by using an upstream specific primer and a first universal primer, wherein the upstream specific primer takes a target sequence complementary to the upstream specific primer as an anchoring site, the first universal primer takes the single nucleotide tail as an anchoring site, and the first universal primer comprises a sequencing primer sequence 1 at the 5 'end and a continuous single base complementary to the single nucleotide tail at the 3' end;

and carrying out second PCR amplification on the product of the first PCR amplification by using a downstream specific primer and a second universal primer, wherein the downstream specific primer is positioned at the downstream of the upstream specific primer and carries a sequencing primer sequence 2 at the 5 'end, and the second universal primer comprises a sequencing primer sequence 1 at the 5' end of the first universal primer.

The method of claim 1, wherein a sequencing tag primer is further added to the second PCR amplification, and the 3 'end of the sequencing tag primer comprises a sequencing primer sequence 2 at the 5' end of the downstream specific primer.

The method of claim 1, further comprising: before adding the single-nucleotide tail, a random base is added to the 3' end of each strand of the single-stranded DNA and/or the double-stranded DNA under the action of terminal transferase.

The method of claim 3, wherein the random bases are 5-10 bases in length.

The method of claim 1, wherein the single polynucleotide tail is 15-30 single bases in length.

The method of claim 1, wherein the upstream specific primer is 25-150bp away from the mutation target site.

The method of claim 1, wherein the first PCR amplification is performed for 10-20 cycles.

The method of claim 1, wherein the contiguous single base is 11-15 contiguous C or G bases, or 25-35 contiguous a or T bases.

The method of claim 1, wherein the first universal primer further comprises a degenerate base after the consecutive single bases.

The method of claim 9, wherein the degenerate base is H, V, B or D.

The method of claim 1, wherein the second PCR amplification is performed for 15-30 cycles.

The method of claim 1, wherein the mutation target sites comprise single nucleotide polymorphisms, insertions and deletions, and copy number variations.

A targeted enrichment kit for detecting low frequency mutations, comprising:

the single-base single-nucleotide tail is added to the 3' end of each strand of the single-stranded DNA and/or the double-stranded DNA under the action of the terminal transferase;

an upstream specific primer and a first universal primer, which are used for carrying out first PCR amplification on a region where a mutation target site is located, wherein the upstream specific primer takes a target sequence complementary to the upstream specific primer as an anchoring site, the first universal primer takes the single nucleotide tail as an anchoring site, and the first universal primer comprises a sequencing primer sequence 1 at the 5 'end and a continuous single base complementary to the single nucleotide tail at the 3' end;

and the downstream specific primer and the second universal primer are used for carrying out second PCR amplification on the product of the first PCR amplification, wherein the downstream specific primer is positioned at the downstream of the upstream specific primer, the 5 'end of the downstream specific primer is provided with a sequencing primer sequence 2, and the second universal primer comprises a sequencing primer sequence 1 at the 5' end of the first universal primer.

The kit of claim 13, further comprising a sequencing tag primer for performing a second PCR amplification on the first PCR amplified product, wherein the 3 'end of the sequencing tag primer comprises a sequencing primer sequence 2 at the 5' end of the downstream specific primer.

The kit of claim 13, further comprising: and (b) mixed nucleotides for adding a random base to the 3' end of the single-stranded DNA and/or each strand of the double-stranded DNA under the action of a terminal transferase before adding the single-nucleotide tail.

The kit of claim 15, wherein the random bases are 5-10 bases in length.

The kit of claim 13, wherein the single polynucleotide tail is 15-30 single bases in length.

The kit of claim 13, wherein the upstream specific primer is 25-150bp away from the mutation target site.

The kit of claim 13, wherein the first PCR amplification is performed for 10-20 cycles.

The kit of claim 13, wherein the contiguous single base is 11-15 contiguous C or G bases, or 25-35 contiguous a or T bases.

The kit of claim 13, wherein the first universal primer further comprises a degenerate base after the contiguous single base.

The kit of claim 21, wherein the degenerate base is H, V, B or D.

The kit of claim 13, wherein the second PCR amplification is performed for 15-30 cycles.

The kit of claim 13, wherein the mutation target sites comprise single nucleotide polymorphisms, insertions and deletions, and copy number variations.