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Probe for nucleic acid enriching capture and design method thereof

A probe and nucleic acid technology, applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve the problems of inability to capture complementary strand nucleic acid libraries, mutation frequency deviation of sequencing results, capture copy number loss, etc. problem, to achieve the effect of improving specificity, increasing original copy number, and improving capture

Active Publication Date: 2016-11-09
AMOY DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The one-way design of the probe makes it impossible to capture the complementary strand nucleic acid library during the capture process, resulting in the loss of capture copy number; in addition, the probe is designed according to the normal genome sequence, which preferentially captures the normal genome sequence instead of the mutant sequence, resulting in mutations in the sequencing results frequency deviation

Method used

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  • Probe for nucleic acid enriching capture and design method thereof
  • Probe for nucleic acid enriching capture and design method thereof
  • Probe for nucleic acid enriching capture and design method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: The effect of bidirectional probes on the onTarget rate of different GC content fragments

[0034] Take 6 genome segments (Homo sapiens, Release 19(GRCh37.p13)

[0035]chr2:29446476-chr2:29446775; chr2:29447226-chr2:29447525;

[0036] chr2:29448065-chr2:29448364; chr6:117642641-chr6:117642940;

[0037] chr6:117645323-chr6:117645622; chr6:117641038-chr6:117641337), carry out probe design: unidirectional probe 3 times overlapping coverage, or bidirectional probe dislocation coverage; probe length is 59bp, probe 3' end Modified for Biotin. There are many specific probe sequences. Taking Homo sapiens, Release 19 (GRCh37.p13) chr2:29446476-chr2:29446775 segment as an example, 5 unidirectional probes are listed, which are SEQ ID NO: 1-5; 5 needles, which are SEQID NO:6-10. Take 30 ng of leukocyte DNA each, build a library with KAPA kit, and use hybridization reagents for 2h and 24h hybridization capture. The hybridization capture uses the SeqCap EZ Hybridizatio...

Embodiment 2

[0042] Example 2, the impact of bidirectional probes on the onTarget rates of different chromosomes

[0043] Take 5 genome segments Homo sapiens, Release 19 (GRCh37.p13) chr2: 29446201-29448364, chr7: 55241635-55241748, chr7: 55242396-55242539, chr7: 55249029-552549107, chr7: 2792 probe as follows; Design: Non-overlapping coverage of unidirectional probes and coverage of bidirectional misplaced probes. The probe length is 35-80bp and the Tm of the probe is 72-78°C, and the 3' end of the probe is modified with Biotin. There are many specific probe sequences. Taking Homo sapiens, Release 19 (GRCh37.p13) chr2: 29446201-29448364 segment as an example, 5 unidirectional probes are listed, which are SEQ ID NO: 11-15; 5 bidirectional probes are listed The bar is SEQ ID NO:16-20, see Table 2. Take 30 ng of leukocyte DNA, build a library with KAPA kit, and use hybridization reagents to perform a hybridization capture for 2 hours. The hybridization capture uses the SeqCap EZ Hybridizat...

Embodiment 3

[0047] Embodiment 3, the influence of bidirectional probe on UID

[0048] Take multiple genome segments for probe design. Multiple genome segments include exon 31, intron 31, and exon 32 of the ROS1 gene and exon 19 of the EGFR gene, etc., and carry out the following scheme Unique probe design: 3-fold overlapping coverage of unidirectional probes, misplaced coverage of bidirectional probes, the length of the probes is 59bp, and the 3' end of the probes is modified with Biotin. After software analysis and test results, probes containing repetitive sequences are eliminated . There are many specific probe sequences, taking exon 19 of the EGFR gene as an example, Homo sapiens, Release 19 (GRCh37.p13) chr7: 55242415-55242513, listed 5 unidirectional probes that are finally applicable to the reference sequence, SEQ ID NO:21-25, enumerates 4 bidirectional probes that are finally applicable to the reference sequence, SEQ ID NO:26-29; taking exon 31, intron 31 and exon 32 of the ROS1 ...

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Abstract

The invention discloses a probe for nucleic acid enriching capture and a design method thereof. A two-way probe consists of a positive-sense strand probe and an antisense strand probe, wherein both the positive-sense strand probe and the antisense strand probe adopt probe zero-lap designs; the lengths of the positive-sense strand probe and the antisense strand probe are 30 to 89 basic groups; the 3' or 5' position of the probe is provided with biotin labels, and can be combined with avidin on magnetic beads. The two-way probe can improve the capture specificity (i.e., the capture on the genome DNA in the non-targeted position can be reduced); the capture on the sample mutant type DNA is obviously improved. The original copy number of the capture sample nucleic acid can be increased.

Description

technical field [0001] The invention relates to the field of gene sequencing, in particular to a probe and design method for nucleic acid enrichment and capture. Background technique [0002] The sequence of the human genome is too large. In order to study the base variation at a specific position in the target gene, it is necessary to enrich the target gene. The more common methods of enrichment are PCR enrichment and hybridization enrichment. PCR enrichment mainly includes multiplex PCR and digital PCR; PCR has limitations such as amplification bias, primer interaction in multiplex amplification, and inability to detect unknown sequences (such as gene recombination). Hybridization enrichment mainly includes chip hybridization and solution hybridization; hybridization is not suitable for small area enrichment and requires a large amount of sample. Chip hybridization is an early technology that promotes the hybridization reaction by increasing the amount of sample; solutio...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
CPCC12Q1/6811C12Q2563/131C12Q2535/122C12Q1/68C12N15/11
Inventor 施伟杰林清华纪斌峰唐郑华李旭超阮力
Owner AMOY DIAGNOSTICS CO LTD
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