Method for enriching nucleic acids with target sequence from nucleic acid sample

A technology of target sequence and nucleic acid sequence, applied in the direction of biochemical equipment and methods, measurement/testing of microorganisms, etc., can solve problems such as no longer suitable

Active Publication Date: 2016-09-07
艾吉泰康(嘉兴)生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Multiplex PCR is more suitable for the capture of hotspot regions or target regions with a small length; for target regions with longer len

Method used

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  • Method for enriching nucleic acids with target sequence from nucleic acid sample
  • Method for enriching nucleic acids with target sequence from nucleic acid sample
  • Method for enriching nucleic acids with target sequence from nucleic acid sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: the design of bait sequence

[0073] 1000 sites on the exons and introns of the human genome were randomly selected (the distribution of these sites is shown in the table) for testing the method of the present invention. Decoy sequences were designed for these 1000 random target sequences for subsequent testing.

[0074] Table 1: Chromosomal distribution of 1000 randomly selected loci

[0075] chromosome

Number

chromosome

Number

chr1

92

chr12

73

chr2

67

chr13

23

chr3

53

chr14

15

chr4

43

chr15

29

chr5

45

chr16

41

chr6

124

Chr17

36

chr7

42

chr18

14

chr8

46

Chr19

31

chr9

34

chr20

21

chr10

61

chr21

9

chr11

80

Chr22

21

[0076] Decoy sequence design includes the following steps:

[0077] 1. First, the target sequence characteristic analysis includes ...

Embodiment 2

[0099] Embodiment 2: Preparation of bait sequence

[0100] Sequence preparation was carried out according to the bait sequence designed in Example 1, and the preparation method of the bait sequence was as follows:

[0101] 1. Add specific sequences with a length of 20 bases to the 5' end and 3' end of the bait sequence respectively. The specific sequence design principles are: 1) No non-specific amplification products will be generated on the target (to be captured) genome ; 2) The GC content is between 30% and 70%, preferably between 40% and 60%; 3) The two will not form dimers, or the free energy of dimers formed is ≤47°C, preferably ≤37°C . Thus forming the sequence to be synthesized, all bait sequences are identical to a pair of specific sequences, examples are as follows:

[0102] 5' specific sequence-bait sequence (60-150bp range)-3' specific sequence is (SEQ ID NO.1):

[0103] ATATAGATGCCGTCCTAGCG-NNNNNNNNNNNNNNNNNNNNNN-TGGGCACAGGAAAGATACTT. Where "NNNNNNNNNNNN...NNNN...

Embodiment 3

[0124] Implementation 3: Target region library capture

[0125] 1. DNA library preparation for high-throughput capture sequencing:

[0126] 1) Take 1 μg of genomic DNA of the tested species, and use the ultrasonic breaker Bioruptor pico to randomly break into small fragments of 150-250bp;

[0127] 2) Use the Illumina TruSeq DNA library preparation kit for pre-capture small fragment library preparation.

[0128] 2. Use the prepared nucleic acid analog pool and the small fragment library of the target species to perform target region library hybridization capture:

[0129] 1) Preparation of blocking primers:

[0130]

[0131] Synthesize according to the above primer sequence, each synthesized at 100 OD, dilute each primer to 1000 μM, mix according to equal volume, and name it Block 1;

[0132] 2) Dilute cot-1 DNA and salmon sperm DNA to 100ng / μl, mix in equal volumes, and mark it as Block 2;

[0133] 3) Mix 6 μl Block 1 and 5 μl Block 2, labeled as Block Mix;

[0134] 4)...

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Abstract

The invention provides a method for enriching nucleic acids with a target sequence from a nucleic acid sample, wherein the method comprises the following steps: providing the nucleic acid sample which contains a the target nucleic acid sequence or a bait sequence which is consistent with the target nucleic acid sequence or has characteristics to the target sequence; conducting in vitro transcription by taking the bait sequence as a template so as to prepare a nucleic acid analogue, wherein the nucleic acid analogue has a binding part; fragmenting the nucleic acid sample; hybridizing the nucleic acid analogue with the nucleic acid sample, so that a nucleic acid analogue/DNA hybrid complex is formed by the nucleic acid analogue and the nucleic acid with the target sequence; and by virtue of the binding part, separating the nucleic acid analogue/DNA hybrid complex from non-specific hybrid nucleic acids, so that the nucleic acids with non-target sequences are removed. According to a preferred embodiment, the method further comprises a step of amplifying the nucleic acid analogue/DNA hybrid complex, so that the purpose of enriching the nucleic acids with the target sequence is achieved.

Description

technical field [0001] The invention relates to the capture, enrichment and analysis of nucleic acid sequences. More specifically, the present invention relates to a method for enrichment of target sequences based on liquid phase capture. Background technique [0002] Whole-genome sequencing can obtain mutations, insertions, deletions, and structural variations at the genome-wide level. However, due to the large capacity of the genome, sequencing at 30× will generate a data volume close to 100G. The low mutation frequency sequencing related to tumors requires at least 1000× coverage, and if the whole genome is sequenced, it will generate as much as 3000G of data. Such a large amount of data will not only cause great difficulties in the analysis of the data, but also make the sequencing cost huge. At this time, the target area capture technology came into being. [0003] The target region capture technology refers to the directional capture of the nucleic acid sequence of...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/68C12Q1/6806C12Q2565/519
Inventor 蔡万世王瑞超屈武斌杭兴宜
Owner 艾吉泰康(嘉兴)生物科技有限公司
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