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A technology of ligase reaction and design method, which is applied in the field of new probe design for ligase reaction, and can solve problems affecting detection results, etc.
Inactive Publication Date: 2015-01-07
宁波有成生物医药科技有限公司
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However, in the traditional MLPA, LCR and other ligase-mediated nucleic acid amplification reactions, due to the l
Method used
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Embodiment 1
[0017] Blood samples from patients with clinically diagnosed non-Hodgkin's lymphoma (NHL) were used as samples. DNA Blood Mini Kit (Qiagen) kit was used to extract blood genomic DNA, and the polymorphic sites rs1800896 and rs1800871 in the promoter region of interleukin 10 were detected by MLPA method.
[0018] According to the probe design method of the present invention, the probe suitable for this detection is designed, and the probe sequence is shown in Table 1:
[0019] Table 1: Probe sequences for detection of rs1800896 and rs1800871
[0020]
[0021] Hybridization: Dissolve the probe in TE to prepare a 5nM solution. Prepare a reaction solution in a PCR tube according to the hybridization reaction system in Table 2, and incubate at 60°C for 16-20 hours for hybridization.
[0022] Ligation: After hybridization, add 3uL of ligase buffer, 0.5uL of ligase, and 6.5uL of ultrapure water into the PCR tube, incubate at 54°C for 20min, heat at 98°C for 5min, and then cool t...
Embodiment 2
[0031] DNA extracted from B lymphocyte LY-01 was used as a sample, and the copy number of gene C1qA was detected by LCR method.
[0032] According to the probe design method of the present invention, the probe suitable for this detection is designed, and the probe sequence is shown in Table 1:
[0033] Table 1: Probe sequences for detecting gene C1qA
[0034]
[0035] LCR reaction: the probe was dissolved in TE to prepare a 5nM solution. Prepare the reaction solution in the PCR tube according to the LCR reaction system in Table 2, place the PCR tube on the PCR instrument, and perform the reaction according to the reaction program in Table 3.
[0036] Table 2: LCR reaction system
[0037]
[0038] Table 3: LCR reaction program
[0039]
[0040] After the LCR reaction, the amplified product was analyzed by capillary electrophoresis. The length of the amplified fragment of the C1qA gene was 100bp. image 3 . Compared with the copy number of the housekeeping gene, in t...
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Abstract
The invention relates to a probe design method and application in ligase-mediated nucleic acid amplification reaction. In the ligase-mediated nucleic acid amplification reaction, one target sequence can be amplified by using 1-2 pairs of probes, and each probe is characterized by comprising a complementary area which is strictly complementary to the target sequence, a filling area (optional) for adjusting the length of each probe, a primer area (optional) used as a PCR primer and an extension area, wherein the length of the extension area is 5-20bp, and the extension area is complementary to a nucleotide sequence of the front end or tail end of the complementary area, namely, the nucleotide sequence of the extension area is determined by the target sequence; when each probe is not combined with the target sequence, the probe is of a stem-and-loop structure; and under a certain condition, a pair of the probes are combined with the target sequence and are connected with the target sequence to form a complete oligonucleotide chain by virtue of the ligase. The probe design method disclosed by the invention can be used for reducing non-specific amplification and avoiding false positive, and is particularly suitable for multiple detection of nucleic acids.
Description
technical field [0001] The invention relates to a probe design method for nucleic acid amplification reaction in the field of molecular biology and medical detection, in particular to a probe design method and application in ligase-mediated nucleic acid amplification reaction. technical background [0002] Multiplex ligation-dependent probe amplification (MLPA) is a new technology for qualitative and semi-quantitative analysis of DNA sequences to be detected, which was first reported by Schouten et al. in 2002. The basic principle of MLPA is: the probe and the target sequence DNA are hybridized, then ligated by ligase, PCR amplified, the product is separated by capillary electrophoresis, and the analysis software analyzes the collected data to draw a conclusion. In an MLPA reaction, amplification of each target sequence requires a pair of probes, each probe consisting of a primer sequence and a specific sequence. After the pair of probes hybridizes with the target sequence,...
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