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A detection system and detection kit for the number of cgg unit repeats in the 5' untranslated region of fmr1 gene

An untranslated region and gene technology is applied in the field of detection systems and detection kits for the number of repeats of CGG units in the 5' untranslated region of the FMR1 gene, and can solve the problems such as the difficulty in determining the maximum product peak that cannot be improved.

Active Publication Date: 2021-09-14
BEIJING MICROREAD GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some studies or patents (such as Chinese patent CN102449171B) add bases that match the specific template sequence at the 3' end of the repeat primer. The main purpose is to more accurately locate the AGG in the CGG repeat region, which cannot improve the Difficulty in determining the largest product peak in repeated products

Method used

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  • A detection system and detection kit for the number of cgg unit repeats in the 5' untranslated region of fmr1 gene
  • A detection system and detection kit for the number of cgg unit repeats in the 5' untranslated region of fmr1 gene
  • A detection system and detection kit for the number of cgg unit repeats in the 5' untranslated region of fmr1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: CGG repeat detection with repeat primers of different lengths complementary to the CGG border sequence

[0062] The following three primers were respectively used as repeat primers to detect the CGG repeat of the sample to be tested:

[0063] Primer A: GCCGCCGCCGCCGCC

[0064] Primer B: AGCCGCCGCCGCCGCC

[0065] Primer C: CCAGCCGCCGCCGCCGCC

[0066] The 3' ends of the three sequences are identical, all of which are complementary to 5 (CGG). The difference between them is that primer A only contains the sequence of the repeat segment without a sequence complementary to the CGG border sequence, which is equivalent to the primer used in the conventional repeat primer PCR (RP PCR); primer B adds 1 to the 5' upstream of the repeat segment bases complementary to the CGG border sequence; Primer C adds 3 bases complementary to the CGG border sequence 5' upstream of the repeat.

[0067] The sequence of the upstream primer used is: FAM-GCCTCAGTCAGGCGCTCAGCTCCGT.

...

Embodiment 2

[0080] In summary, it is difficult to determine the maximum product peak simply by using a repeated primer (repeated primer A); using a repeated primer with a fragment complementary to the CGG border sequence at the 3' end can increase the peak height of the maximum product peak, so that the maximum product peak can be accurately determined by cleaning. Product peak; as a preference, the repeat primer B with a matching base added at the 3' end of the repeat fragment has the best discrimination effect. Embodiment 2: Using the detection kit disclosed in the present invention to detect different types of samples

[0081] Kit components include: enzyme mixture, full-length primer mixture, duplicate primer mixture, amplification buffer, positive control, sterile water, internal standard and other components.

[0082] Use the kit to detect three samples to be tested.

[0083] The specific detection steps are as follows:

[0084] 1) Prepare the PCR amplification reaction system. E...

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Abstract

The invention discloses a detection system and a detection kit for the number of CGG unit repeats in the 5' untranslated region of a gene. The detection system includes 3 primers located upstream, downstream and at the border of the CGG repeat fragment, and the number of CGG repeat units can be judged according to the two results of the detected full-length product size and the number of CGG products. In particular, since the primers located at the border of the repeat segment have a stronger binding ability to the corresponding template, the largest CGG product can be determined more clearly, thereby accurately determining the specific number of CGG repeats, and can effectively and accurately determine the number of repeats less than 60. Repeat number, and can clearly determine whether there is a genotype with a larger repeat number.

Description

technical field [0001] The invention relates to the detection of gene CGG unit repeat quantity, which can provide reference for clinical diagnosis of fragile X chromosome syndrome, and belongs to the clinical molecular detection technology in the field of biomedicine. Background technique [0002] Fragile X Syndrome (FXS) is a common X-linked genetic disease. The typical main symptoms are moderate to severe mental retardation, accompanied by abnormal behavior and physical development. Its incidence is second only to Down's syndrome in hereditary mental retardation syndrome, accounting for 10-20% of male mental retardation and 40% of X-linked mental retardation. [0003] The occurrence of fragile X syndrome is closely related to the abnormality of FMR1 gene. More than 95% of fragile X syndromes are caused by CGG repeat expansion mutations in the 5' untranslated region of the X chromosome FMR1 gene, and less than 5% are caused by missense and deletion mutations in the FMR1 ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6883C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 李琛张晔何梦娟邵苗苗陈初光
Owner BEIJING MICROREAD GENE TECH