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Application of bridge type oligonucleotides to library target region capturing

A technology of sequence capture and target sequence, which is applied in chemical library, microbiological determination/testing, combinatorial chemistry, etc. It can solve the problems of affecting capture rate, poor tag sequence blocking effect, and high cost of synthesizing hypoxanthine, reaching the scope of application wide effect

Active Publication Date: 2018-10-19
IGENETECH BIOTECH (BEIJING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to this order of magnitude, the amount of cost required to synthesize a closed sequence is extremely inoperable
In addition, closed sequences corresponding to 96 kinds of tag sequences were added in the process of building the library, which increased the cumbersomeness of the experimental operation in the process of building the library
[0003] In order to control the cost, it was also proposed that a corresponding number of hypoxanthine be used to block the tag sequence. However, hypoxanthine has a certain preference for the blocked base, resulting in poor blocking effect on some tag sequences. This affects the capture rate, and the cost of synthesizing hypoxanthine is relatively expensive

Method used

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  • Application of bridge type oligonucleotides to library target region capturing
  • Application of bridge type oligonucleotides to library target region capturing
  • Application of bridge type oligonucleotides to library target region capturing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1-Illumina sequencing platform library construction process:

[0066] 1. Library structure and closed oligonucleotide structure

[0067] 1) Illumina sequencing platform prePCR library structure: For clarity, two chains are shown, the structure of the two sequences is as follows:

[0068] a (5' to 3'):AATGATACGGCGACCACCGAGATCTACAC (SEQ ID NO.1) [tag sequence] ACACTCTTTCCCTACACGACGCTCTTCCGATCT (SEQ ID NO.2) [pairing sequence with target sequence] AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC (SEQ ID NO.3) [tag sequence]CTATCTTGTATGTG SEQ ID NO. 4)

[0069] b (3' to 5'): TTACTATGCCGCTGGTGGCTCTAGATGTG (SEQ ID NO.5) [tag sequence]TGTGAGAAAGGGATGTGCTGCGAGAAGGCTAGA (SEQ ID NO.6) [pairing sequence with target sequence] TCTAGCCTTCTCGTGTGCAGACTTGAGGTCAGTG (SEQ ID NO.7) [tag sequence]TAGAGACCATACGG SEQ ID NO. 8)

[0070] The length of the paired sequence with the target sequence is 80-120 bp, preferably 100 bp.

[0071] 2) Blocked oligonucleotide sequence of Illumina sequencing platform:

[00...

Embodiment 2

[0195] Example 2-NEB sequencing platform library construction process

[0196] 1. Library structure and closed oligonucleotide structure

[0197] 1) The structure of the prePCR library of NEB sequencing platform: For clarity, the structure of the front and back strands and the double-stranded two sequences are shown as follows:

[0198] a(5’ to 3’):

[0199] CAAGCAGAAGACGGCATACGAGAT(SEQ ID NO.9)[Tag sequence]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(SEQ ID NO.10)[pairing sequence with target sequence]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT(SEQ ID NO.14)

[0200] b(3’ to 5’):

[0201] GTTCGTCTTCTGCCGTATGCTCTA(SEQ ID NO.15)[Tag sequence]CACTGACCTCAAGTCTGCACACGAGAAGGCTAGA(SEQ ID NO.16)[pairing sequence with target sequence]TCTAGCCTTCTCGCAGCACATCCCTTTCTCACATCTAGAGCCACCAGCGGCATAGTAA (SEQ ID NO.17)

[0202] The length of the paired sequence with the target sequence is 80-120 bp, preferably 100 bp.

[0203] 2) NEB sequencing platform blocking oligonucleotide sequence:

[0204] a-1: AGA...

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Abstract

The invention discloses an improved sequence capturing method, which comprises the steps of 1, extracting DNA; breaking the DNA; 2, connecting the two ends of the broken DNA with a connector, whereinthe remote end of a connector sequence at one end of the broken DNA includes a tag sequence with the length being 6 to 8nt; 3, performing Pre-PCR on the DNA of the connecting connector; 4, mixing a probe and a sealing reagent; capturing the Pre-PCR, wherein the sealing reagent includes sealing sequences complementary with sequences except the broken DNA, and the tag sequences are sealed by C3 spacer arms with the same length; 5, after the hybrid capture, performing PCR amplification. A bridge type sealed design strategy is used; corresponding sealing sequences are respectively designed for connector sequences at two ends of inserting fragments; the tag sequences at the middle part are subjected to bridge type connection by the C3 spacer arms; the cost is reduced; the complexity in the library building process is simplified.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses the application of a bridge oligonucleotide in capturing target regions of libraries. Background technique [0002] During the library construction process, some sequences other than the inserted fragments are often introduced, and these sequences are of great significance for on-machine sequencing, sample differentiation, and tracing the origin of the original DNA molecules. However, during the hybridization capture process, non-target sequences other than these inserted fragments need to be effectively blocked. The significance of blocking these sequences is to prevent non-specific binding of the probe to sequences outside these target regions during the hybridization process. At the same time, the tag sequence also needs to be effectively blocked, otherwise libraries with different inserted fragments will anneal to the tag sequence, causing a large nu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2531/113C12Q2525/186
Inventor 蔡万世余越美梁加龙王瑞超邵谦之杭兴宜
Owner IGENETECH BIOTECH (BEIJING) CO LTD
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