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PCR detection kit for CTG region of atrophic myotonin protein kinase gene and application of PCR detection kit

A technology of protein kinase gene and detection kit, which is applied in the field of PCR amplification, can solve the problems of unsuitable for routine diagnosis and complicated operation, and achieve the effect of low cost, easy operation and good application prospect

Active Publication Date: 2022-02-22
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

SP-PCR and FSP-PCR are complex and unsuitable for routine diagnostic applications

Method used

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  • PCR detection kit for CTG region of atrophic myotonin protein kinase gene and application of PCR detection kit
  • PCR detection kit for CTG region of atrophic myotonin protein kinase gene and application of PCR detection kit
  • PCR detection kit for CTG region of atrophic myotonin protein kinase gene and application of PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The design of embodiment 1 specific primer pair

[0030] (1) The main purpose of this embodiment is to select a primer pair with higher specificity for the amplification of the CTG repeat region of the 3'-UTR of the DMPK gene. The DNA samples are all homozygous allele samples without CTG expansion. If the amplification result is not a single band, it shows that the amplification specificity of the primers is not very good.

[0031] Through comparative analysis of the following documents, two pairs of primers were selected as shown in Table 1. Then use these two pairs of primers to carry out PCR amplification on the three DNA samples respectively.

[0032] Document 1 is: Novel Heat Pulse Extension-PCR Based Method for Detection of Large CTG-Repeat Expansions in Myotonic Dystrophy Type 1

[0033] Document 2 is: (CTG)n Expansion at DMPK locus seen only in muscle tissue: Anovel case

[0034] Table 1 Primer names and sequence information

[0035]

[0036] (2) Reaction...

Embodiment 2

[0045] Example 2 No / Low Preference Experimental Condition Screening

[0046] The main purpose of this example is to achieve no / low bias amplification of the CTG repeat region of the 3'-UTR of the DMPK allele by optimizing different experimental conditions and using the above-mentioned specific primer pair 1. DNA samples included 2 homozygous allele samples without CTG expansion and 1 heterozygous allele sample with CTG repeat expansion. The expected PCR result is a single band for 2 homozygous allele samples without CTG expansion, and two bands of different sizes for 1 heterozygous allele sample with CTG repeat expansion.

[0047] The DNA samples S4, S5, and S6 in this embodiment are respectively: S4 and S5 are normal human blood DNA samples; S6 is a DM1 patient blood DNA sample.

[0048] 1. Experimental conditions

[0049] (1) The reaction system and reaction conditions of Condition 1 are shown in Table 4 and Table 5 respectively.

[0050] Table 4 reaction system

[0051]...

Embodiment 3

[0088] Example 3 The amplification result of condition 6 is verified by first-generation sequencing

[0089] In order to further confirm that the larger band in S6 amplified by condition 6 is the target band, a first-generation sequencing was performed on it, and the results are as follows Figure 4 and Figure 5 shown.

[0090] Figure 4 The results showed that there was a CTG repeat sequence in the forward sequencing results, and the specific sequence at the 5' end was also consistent; Figure 5 The results showed that there was a CAG (CTG reverse complementary sequence) repeat sequence in the reverse sequencing results, and the specific sequence at the 5' end was also consistent; analysis showed that the larger fragment in the S6 lane of condition 6 was the target band.

[0091] Example verification of embodiment 4 amplification preference

[0092] The first purpose of this embodiment is to carry out PCR amplification with the reaction system and amplification program o...

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Abstract

The invention provides a PCR detection kit for a CTG region of an atrophic myotonic protein kinase gene, the PCR detection kit comprises a reaction system, the reaction system comprises LongAmp Taq DNA polymerase, 5x GC buffer, dNTPs, a DNA sample, water and a pair of specific primer pairs, and nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2. The method has the characteristics of strong amplification specificity and small amplification preference. The invention also provides an application of the PCR detection kit for the CTG region of the atrophic myotonic protein kinase gene. The PCR detection kit provided by the invention can be used for amplifying a hybrid allele sample carrying CTG repeated expansion, and can be used for detecting the number of CTG tribasic group repetition times of an atrophic myotonic protein kinase gene 3 '-UTR at the same time.

Description

technical field [0001] The invention belongs to the technical field of PCR amplification, and in particular relates to a PCR detection kit for the CTG region of atrophic myotonic protein kinase gene and its application. Background technique [0002] Myotonic Dystrophy Type 1 (DM1) is a genetic disease characterized by muscle weakness, muscle rigidity, and muscle atrophy, accompanied by endocrine, heart, eye lens and other system damage. DM1 is caused by the expansion of the CTG three-base repeat in the 3'-UTR region of the DMPK gene located on chromosome 19q13.2-13.3. Generally, the number of CTG repeats is greater than 50 to cause disease; the age of onset and the number of CTG repeats are negatively correlated. And different CTG repeat times have different main complications and their severity. With the inheritance of the disease-causing gene, under normal circumstances, the number of CTG repeats in the DMPK gene of the next generation is more than that of the previous ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2565/125
Inventor 罗苏珊吴群峰朱雯华奚剑英林洁杜雪柯严陈燕
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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