Method for detecting the mutation and methylation of tumor-specific genes in ctDNA

A method for detecting target mutation and/or methylation in a DNA sample, comprising the following steps:

• (1) constructing a library;
• (2) performing two rounds of nested PCR amplification to the library obtained in step (1), sequencing the product, and analyzing the occurrence of target mutation and/or methylation in the DNA sample according to the sequencing result;
• in the step (2), primer combination A is used to carry out the first round of PCR amplification;
• primer combination A consists of upstream primer A and downstream primer combination A;
• the upstream primer A is a library amplification primer used for library amplification in step (1);
• the downstream primer combination A is a combination of Y primers designed according to X target sites;
• X and Y are both natural numbers greater than 1, and X≤Y;
• using the product of the first round of PCR as a template, carrying out the second round of PCR amplification with primer combination B;
• primer combination B consists of upstream primer B, downstream primer combination B and index primer;
• the upstream primer B is a library amplification primer and the 3′ end is the same as that of the upstream primer A, and is used for the amplification of the product of the first round of PCR;
• the index primer includes a segment A for sequencing, an index sequence for distinguishing samples, and a segment B for sequencing from the 5′ end;
• the primer in the downstream primer combination B has the segment B and form a nested relationship with the primer detecting the same target site in the downstream primer combination A;
• wherein, the method for constructing a library in step (1) comprises the following steps sequentially:
• 1) taking a DNA sample and digesting it with a methylation-sensitive restriction endonuclease;
• 2) the DNA sample digested in step 1) is subjected to end repair and adding A treatment at the 3′ end sequentially;
• 3) ligating the DNA sample processed in step 2) with an adapter in an adapter mixture, and obtaining a library after PCR amplification;
• 4) amplifying the library obtained in step 3);
• the adapter mixture consists of n adapters;
• each adapter is formed by an upstream primer A and a downstream primer A to form a partial double-stranded structure;
• the upstream primer A includes a sequencing adapter A, a random tag, an anchor sequence A and a base T sequentially from the 5′ end;
• the downstream primer A includes an anchor sequence B and a sequencing adapter B sequentially from the 5′ end;
• the partial double-stranded structure is formed by the reverse complementation of the anchor sequence A and the anchor sequence B;
• the sequencing adapter A and sequencing adapter B are corresponding sequencing adapters selected according to different sequencing platforms;
• the random tag is 8-14 bases in length;
• the anchor sequence A has a length of 12-20 bp, and has ≤3 consecutive repeating bases;
• the n adapters use n different anchor sequences A(s), and the four bases in each anchor sequence A are balanced, and the number of mismatched bases ≥3;
• n is any natural number ≥8;
• a primer pair used for amplification in step 4) consists of two single-stranded DNA molecules shown in SEQ ID NO. 25 and SEQ ID NO. 26 in the sequence listing.