Method and primers for detecting mutation of NOP10 gene exon 2 mutation site R34W(C100T) sequence

A technology of NOP10-R and NOP10-F, which is applied in the fields of life science and biology, can solve the problems of single and low abundance of non-specific amplification products, and achieve the effect of simple operation, low cost and improved amplification specificity

Inactive Publication Date: 2017-01-04
北京艾迪康医学检验实验室有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Touch-down PCR amplification can ensure that the combination of the forward and reverse amplification primers and the sample DNA template occurs between the most complementary sequences. When the annealing temperature is reduced to the level where non-specific amplification occurs, the specific amplification product is At this time, there is already a geometric number of initial advantages, and the abundance is high. In the remaining amplification reactions, the specific amplification products compete with the non-specific amplification products, but due to the low abundance of non-specific amplification products , the specific amplification product is always preferentially amplified, resulting in a single dominant specific amplification product

Method used

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  • Method and primers for detecting mutation of NOP10 gene exon 2 mutation site R34W(C100T) sequence
  • Method and primers for detecting mutation of NOP10 gene exon 2 mutation site R34W(C100T) sequence
  • Method and primers for detecting mutation of NOP10 gene exon 2 mutation site R34W(C100T) sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The primers for detecting the whole exon sequence of the No. 2 exon of the NOP10 gene include: forward and reverse primers for amplifying the whole exon sequence of the No. 2 exon of the NOP10 gene; its base sequence is:

[0041] NOP10-F: TGTAAAACGACGGCCAGTTTGACCATTGCATCTTCTATTTTC

[0042] NOP10-R: AACAGCTATGACCATGTCCCCTTTATGGGTGATGCCAC.

[0043] Preferably, the primers also include a pair of sequencing primers, the base sequence of which is

[0044] M13-F: TGTAAAACGACGGCCAGT

[0045] M13-R: AACAGCTATGACCATG.

[0046] In the detection, first use the above forward and reverse primers to amplify the whole exon sequence of exon No. 2 of the NOP10 gene to obtain the amplified product, then use the above pair of sequencing primers to sequence the amplified product to obtain the amplified The gene sequence of the augmented product.

[0047] The kit for detecting the whole exon sequence of exon No. 2 of NOP10 gene, including: sample DNA extraction reagent (for example, use t...

Embodiment 2

[0060] Detection process:

[0061] (1) Use the blood DNA extraction kit (Tiangen Biology) to extract the genomic DNA in the blood sample:

[0062] 1) Take 500uL of blood and add 1000uL of red blood cell lysate, mix by inversion, and let stand at room temperature for 5 minutes, during which time, invert and mix several times. Centrifuge at 3000rpm for 5min, suck off the supernatant, leave the white blood cell pellet, add 200uL buffer GA, shake until thoroughly mixed.

[0063] 2) Add 20 μl proteinase K solution and mix well.

[0064] 3) Add 200 μl of buffer GB, mix thoroughly by inversion, place at 70°C for 10 minutes, the solution should become clear, and briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0065] 4) Add 200 μl of absolute ethanol, vortex and mix well for 15 seconds. At this time, flocculent precipitates may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cap.

[0066] 5) Put the solution and floc...

Embodiment 3

[0091] The nucleic acid detection kit of the present invention is used to detect clinical samples.

[0092] 20 anticoagulant blood samples from patients with dyskeratosis congenita were taken for inspection, and the genomic DNA in the samples was extracted according to the detection process described in Example 2, and reagents were prepared and tested.

[0093] Add 2 ul of the genomic DNA solution of each sample extracted according to the detection process described in Example 2 into the PCR reaction solution of the detection system. Do positive, negative and blank controls at the same time. Detect with common PCR instrument, the time is 160 minutes.

[0094] Electrophoresis results such as figure 1 As shown, it shows that the primers NOP10-F and NOP10-R of the present invention can effectively amplify blood samples, and the band is single.

[0095] The NOP10 gene R34W forward sequencing results of sample 1 are as follows: figure 2 As shown, it is wild type, and no R34W m...

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Abstract

The invention discloses a method and primers for detecting mutation of the NOP10 gene exon 2 mutation site R34W(C100T) sequence. The primers include the forward and reverse primers for amplifying the NOP10 gene exon 2 mutation site R34W(C100T) sequence and a pair of sequencing primers. By combining Touch-down PCR amplification and Sanger sequencing, the mutation of the NOP10 gene exon 2 mutation site R34W(C100T) sequence in the body of a patient suffering from dyskeratosis congenita can be rapidly detected.

Description

technical field [0001] The invention belongs to the field of life science and biotechnology, and particularly relates to a primer, a method and a kit for detecting the sequence of the No. 2 exon mutation site R34W (C100T) of the NOP10 gene. Background technique [0002] Dyskeratosis congenita (dskeratosis eongenita, DC) is a bone marrow failure syndrome with genetic heterogeneity, with an incidence of about 1 / 1 million. About 80% to 90% of typical DC patients have the triad of abnormal skin and mucous membranes, which are manifested as reticular pigmentation of the skin, atrophy of the finger (toe) nails, and leukoplakia of the oral mucosa. At present, the mutated genes reported in the literature that can cause DC mainly include CTC1, DKC1, TERC, TERT, TINF2, NHP2, NOP10 and WRAP53. It has been reported in the literature that these genes have three modes of inheritance, which are X-linked recessive inheritance, autosomal recessive inheritance, and autosomal dominant inherit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2535/101C12Q2527/101
Inventor 单战林有升王淑一
Owner 北京艾迪康医学检验实验室有限公司
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