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dna tag sequence and sequencing library construction method and kit

A technology for labeling sequences and sequencing libraries, applied in the field of molecular biology, can solve the problems of DNA library sample loss, cumbersome operation process, and many steps of library construction, and achieve the goals of avoiding uncertain factors, simple steps, optimizing sensitivity and repeatability Effect

Active Publication Date: 2018-05-22
SUREXAM BIO TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, there are some shortcomings in the current method of preparing tagged libraries required for next-generation sequencing: the existing sequencing platforms have many and complicated steps for library construction, each step is prepared independently, and needs to be purified separately to ensure that there is no gap between each step. It will interfere with and contaminate each other, resulting in the inevitable loss of DNA library samples during the purification process, and the operation process is cumbersome

Method used

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  • dna tag sequence and sequencing library construction method and kit
  • dna tag sequence and sequencing library construction method and kit
  • dna tag sequence and sequencing library construction method and kit

Examples

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Comparison scheme
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Embodiment 1

[0044] Embodiment 1 A kind of DNA tag

[0045] In this example, on the basis of satisfying the above DNA tag design conditions, three sets of DNA tags with high specificity are designed, as shown in Table 1, Table 2, and Table 3, wherein the DNA tag sequence in Table 1 is 6 bases , the DNA tag sequence in Table 2 is 7 bases, and the DNA tag sequence in Table 3 is 8 bases, wherein, the PCR amplification primers of the same sequencing reaction system are selected from the DNA tags in the same list, and are combined with PCR The amplification primers are combined to form corresponding tag amplification primers.

[0046] Table 1 DNA tag sequence (6bp)

[0047] SEQ ID NO.

Tag sequence (5'→3')

SEQ ID NO.

Tag sequence (5'→3')

1

TAGCCA

11

GATACG

2

ACATGC

12

GTACAG

3

TGTGCA

13

GACTGT

4

ACGTTG

14

AGTGCT

5

GAGCTA

15

TACCAG

6

GTCTGA

16

TTCGCA

7

CAGACT

1...

Embodiment 2

[0052] Example 2 Indexing PCR primers, kits for sequencing libraries constructed by PCR primer pairs and DNA tags

[0053] 1. PCR amplification primers

[0054] This example takes the detection of EGFR gene and BRAF gene mutations as an example, using the DNA tag sequence described in Example 1 and the amplification primers for the EGFR gene and BRAF gene mutation sites detected by the target to construct a sequencing library kit. That is to construct specific label PCR primers, according to the method of the present invention to determine whether there are mutations in the target detection genes of various DNA samples.

[0055] The present invention designs PCR primers for EGFR gene and BRAF gene, utilizes this set of PCR primers to carry out PCR amplification on the DNA sample, and can amplify target detection gene fragments by one-step PCR, and the specific PCR primers are:

[0056] Table 4 PCR amplification primers

[0057]

[0058] 2. Labeling PCR primers and DNA lab...

Embodiment 3

[0066] Example 3 uses the labeled PCR primers in Example 2 to detect samples

[0067] 1. Sample DNA extraction:

[0068] Refer to the instructions of the AxyPrep Whole Blood Genome Mini Extraction Kit to obtain the DNA to be detected.

[0069] 2. PCR amplification of samples to be tested

[0070] Prepare labeling primer working solution: according to the order of 20 samples in Table 5, take 100ul of PCR primer stock solution with DNA labeling in 1.5ml microcentrifuge tubes, assemble into 20 tubes of labeling PCR amplification primers, and mix well. Multiplex PCR primer working solution, for each sample, each tube of working solution contains amplification primers for EGFR gene and BRAF gene with specific DNA tags introduced at the 5' end. Amplify the target sequence containing the mutation site respectively, and the PCR reaction system is as follows:

[0071]

[0072] The PCR amplification program is: 95°C for 3min; 94°C for 20s, 56°C for 30s, 72°C for 30s, 30 cycles; 72...

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Abstract

The invention relates to a DNA tag and a pair of PCR primers connected to the DNA tag at the 5' end. The invention also provides a method and a kit for constructing a sequencing library consisting of the DNA tag and the pair of PCR primers. The DNA tag provided by the present invention and the amplification primer form a detection product that achieves optimization and balance between specificity, sensitivity and repeatability, and can detect 1-20 samples from different sources at a time, and can accurately distinguish samples from various sources. For the base sequence, the coincidence rate between the high-throughput sequencing described in the present invention and the sequencing method is as high as 100%.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a method for constructing a DNA tag sequence and a sequencing library and a kit. technical background [0002] As an important experimental technique, DNA sequencing has been widely used in biological research. In 1977, Sanger invented the landmark end-termination sequencing method. Because the Sanger method is simple and fast, and has been continuously improved, it has become the mainstream of DNA sequencing (ie, generation sequencing) so far. As the main research method and gold standard of genome research, next-generation sequencing has made great achievements in the past few decades, and has also made clinical molecular diagnosis possible, but it is costly, time-consuming, and low-throughput. It is widely used in clinical practice. With the completion of the Human Genome Project, people have entered the post-genome era, that is, the er...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C40B40/06C40B50/06C12Q1/6869
Inventor 刘志明吴诗扬廖传荣
Owner SUREXAM BIO TECH
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