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Probe preparation method for multi-gene capture sequencing

A multi-gene and probe technology, applied in biochemical equipment and methods, DNA preparation, microbial measurement/inspection, etc., can solve problems such as expensive, difficult to promote, and high cost

Inactive Publication Date: 2015-07-22
张道允 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

RNA probes are inconvenient to transport and store, and are more expensive than DNA
IDT’s DNA probes are synthesized by column synthesis and then mixed together instead of chip synthesis. Compared with the amount of whole exons, the cost of synthesis is also extremely expensive, and it is extremely difficult to promote.
Although Agilent's and IDT's probes are single-stranded and nearly identical in terms of the driving force for hybridization, they are both extremely expensive

Method used

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  • Probe preparation method for multi-gene capture sequencing
  • Probe preparation method for multi-gene capture sequencing
  • Probe preparation method for multi-gene capture sequencing

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preparation example Construction

[0061] Please see attached figure 1 and attached figure 2 As shown, a method for preparing probes for multi-gene capture sequencing, the method at least includes the following steps:

[0062] Step 1: Prepare probes.

[0063] Step 2: Capture target DNA.

[0064] Step 3: Carry out the elution procedure.

[0065] Step 4: Use a high-fidelity enzyme reaction amplification system to amplify after capturing the target DNA.

[0066] Step 5: After the amplified product is purified with magnetic beads, it is quantified to 20 ng / ul with Nanodrop, and stored in aliquots.

[0067] Step 6: Use the Real-time PCR method to detect the positive control sequence to verify the capture efficiency.

[0068] In said step 1, at least the following sub-steps are included:

[0069] Step 1.1: Synthesize 12K, 60K, 90K and other chips with different oligonucleotide quantities. The chips contain a 120bp target region and a common 15-base end:

[0070] 5'-ATCGCACCAGCGTGTN120CACTGCGGCTCCTCA-3'.

[0...

Embodiment 1

[0115] Preparation of capture probes targeting cancer-related genes. A mixture of 120bp+30bp fragment lengths of 319 related genes (as shown in Supplementary Table 1) was prepared in total.

[0116] Step 1: Prepare probes.

[0117]Step 1.1: Synthesize chips with 12K, 60K, 90K and other different oligonucleotide quantities, these chips contain 120bp target regions and common 15 base ends.

[0118] 5′-ATCGCACCAGCGTGTN120CACTGCGGCTCCTCA-3′

[0119] Step 1.2: Dissolve the synthesized oligonucleotide library in 400ul low concentration TE (10mM Tris-HCL, pH8, 0.1mM EDTA).

[0120] Step 1.3: Since the concentration of each oligonucleotide is at the fmol level, amplification is required to obtain the desired concentration.

[0121] Primer sequence: A 5′Biotin-CTGGGAATCGCACCAGCGTGT-3′

[0122] B 5′-CGTGGATGAGGAGCCGCAGTG-3′

[0123] Amplification conditions: (prepare three 50ul PCR mixes), templates are 1ul, 2ul, 5ul,

[0124]

[0125]

[0126] The PCR reaction conditions ar...

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Abstract

The invention discloses a probe preparation method for multi-gene capture sequencing. The method at least comprises the following steps: step 1, preparing a probe; step 2, capturing target DNA; step 3, performing an elution program; step 4, performing post-amplification on the captured target DNA by using a high-fidelity enzyme reaction amplification system; step 5, purifying the amplification product by using magnetic beads, quantifying to 20ng / ul by using Nanodrop, split charging and preserving; and step 5, detecting a positive control sequence by using a Real-time PCR method, and verifying the capture efficiency. A single-chain DNA capture probe is synthesized by utilizing a chip technology and comprises a single-chain biotin-labeled oligonucleotide probe pool obtained by DNA chip synthesis, probe cutting, nucleic acid amplification and DNA melting.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing probes for multi-gene capture sequencing. Background technique [0002] In recent years, research based on next-generation sequencing technology has developed rapidly. In the field of next-generation sequencing technology, the second-generation sequencing technology represented by Illumina, Agilent, Roche, and ABI has greatly reduced the cost of sequencing. occupies an important position. The biggest feature of second-generation sequencing technology is high throughput, which can simultaneously sequence hundreds of millions of DNA fragments. Although high-throughput sequencing technology has greatly reduced the cost of sequencing, it still costs thousands of dollars to sequence a person's entire genome. For projects that require a large number of clinical samples for disease research, the total sequencing cost is still high. More importantly, the huge data vo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10C12N15/11
Inventor 张道允巩子英
Owner 张道允
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