阅读说明：本技术 一种用于测定fmr1基因cgg重复序列中agg插入数量和位置的试剂盒及其使用方法 (Kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene and using method thereof ) 是由 赵立明 姜莹玉 于 2020-12-10 设计创作，主要内容包括：本发明公开了一种用于测定FMR1基因CGG重复序列中AGG插入数量和位置的试剂盒及其使用方法,该试剂盒包括引物群、PCR反应液、针对CGG序列的高效DNA聚合酶、FMR1基因前突变对照品、FMR1基因灰区对照品、FMR1基因正常型对照品、ROX标记和去离子水。本发明所述的试剂盒可用于高效地扩增FMR1基因5’端的CGG重复序列,并利用毛细管电泳仪对CGG重复序列中AGG数目的数量和位置进行准确判读；可以用于快速准确地查出FMR1前突变携带者CGG序列中的AGG插入的位置,更加客观而且准确地对FMR1基因前突变携带者的生育风险进行评估。(The invention discloses a kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene and a using method thereof, wherein the kit comprises a primer group, PCR reaction liquid, efficient DNA polymerase aiming at the CGG sequence, an FMR1 gene pre-mutation reference substance, an FMR1 gene gray area reference substance, an FMR1 gene normal type reference substance, ROX marker and deionized water. The kit can be used for efficiently amplifying the CGG repetitive sequence at the 5' end of the FMR1 gene, and accurately interpreting the quantity and the position of the AGG number in the CGG repetitive sequence by using a capillary electrophoresis apparatus; can be used for quickly and accurately finding out the AGG insertion position in the CGG sequence of the FMR1 pre-mutation carrier, and more objectively and accurately evaluating the fertility risk of the FMR1 pre-mutation carrier.)

1. A kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene is characterized by comprising a primer group, PCR reaction liquid, DNA polymerase, FMR1 gene pre-mutation control, FMR1 gene normal control, ROX marker and deionized water;

the primer group comprises an upstream primer and a downstream primer, the sequence of the upstream primer is CGGCGGCGGCGGCGAA and is shown as SEQ ID NO. 1, the sequence of the downstream primer is ACCAGCTCCTCCATCTTCTCTTCAG and is shown as SEQ ID NO. 2, and the 5' end of the downstream primer R is marked with a fluorescent probe.

2. The kit of claim 1, wherein the ROX marker consists of 20 DNA fragments of different sizes: less than 100bp, 1; 4 pieces of 100bp-200 bp; 201bp-300bp, 2 strips; 3 pieces of 301bp-400 bp; 401bp-500bp, 2 strips; 501bp-600bp, 3 strips; 601bp-700bp, 1 strip; 701bp-800bp, 2 strips; 901bp-1100bp, 2 pieces.

3. The kit according to claim 2, wherein the GC content of all sequences in the ROX marker is 80% or more.

4. The kit according to claim 1, wherein the PCR reaction solution comprises: Tris-HCl, DMSO, betaine or its analogues, GC-melt, magnesium chloride, UNG enzyme and dNTPs.

5. The kit of claim 1, wherein the FMR1 pre-mutation control comprises a DNA sequence equal to 100 CGG repeats in length; the FMR1 gene normal control comprises a DNA sequence equal to 30 CGG repeats in length.

6. The kit of claim 1, wherein the deionized water is autoclaved deionized water.

7. A method of using the kit of any one of claims 1 to 6, comprising the steps of:

(1) extracting human cell genome DNA, ultraviolet light-splitting and quantifying, OD₂₆₀nm/OD₂₈₀The ratio of nm should be between 1.8-2.0; OD₂₆₀nm/OD₂₃₀The ratio of nm should be greater than 2.0, using deionized water to dilute the genomic DNA concentration to 15-50 ng/. mu.L, then placing 2. mu.L in each PCR tube;

(2) adding 2 μ L of FMR1 gene normal control and FMR1 gene pre-mutation control into parallel PCR tubes;

(3) and (3) PCR amplification: mixing a detected DNA sample, an FMR1 gene full-mutation quality control product, an FMR1 gene normal quality control product, the primer group, DNA polymerase and PCR reaction liquid to form a PCR reaction system, wherein the final concentration of the primers in the PCR reaction is 750nM, the total reaction system is 15 mu L, and the reaction system comprises the following components:

11.45. mu.L of PCR buffer solution,

the primer is 3.0 mu L in volume,

0.05. mu.L of DNA polymerase,

sample DNA, FMR1 pre-mutation control or FMR1 gene normal control, 1.0 μ L;

the PCR reaction can be carried out on a conventional PCR instrument, and the amplification procedure is as follows:

circulating for 1 time at 98 ℃ for 5 minutes;

circulating for 30 times at 97 ℃, 35 seconds, 60 ℃, 35 seconds, 68 ℃ and 4 minutes;

circulating for 1 time at 72 ℃ for 10 minutes;

at 4 ℃, keeping constant, and circulating for 1 time;

(4) fragment analysis: the PCR products were mixed with ROX marker, HiDi formamide in the following proportions and analyzed using a capillary electrophoresis apparatus: Hi-Di formamide 10 μ L, ROX labeled 1 μ L, PCR product, 3 μ L;

(5) according to the fragment analysis result of the gene analyzer, selecting the Peak with the highest signal value as an analysis object, and automatically recording the molecular size of the analysis object by the system, wherein the mark is Peak_iTo Peak_iSubstituting into the following formula to calculate to obtain CGG_iValue of (a), theoretically c₀The value of 83.2, m₀The value of the carbon dioxide is 2.92,

，

in the formula, CGG_iThe number of repeats of the CGG sequence following AGG is considered to be the number of repeats of the CGG sequence since AGG is generally present at positions 9-10 of the 5' end of the CGG sequence_i+10 "is greater than the shorter total length of CGG, the AGG is located in the longer pre-mutant sequence, and vice versa in the shorter wild-type CGG sequence.

Technical Field

The invention relates to the technical field of biological engineering, in particular to a kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene and a using method thereof.

Background

Fragile X syndrome (OMIM #30955) is the second largest intellectual impairment disorder disease with a second incidence next to down syndrome, and is also the most common genetic disorder leading to male intellectual impairment. The Fragile X Mental Retardation No. 1 gene (FMR 1) located on the long arm of the X chromosome has a triplet nucleotide repeat sequence in CGG units at the 5' end of the noncoding region. As the number of CGG repeats increases, the patient may exhibit a range of clinical symptoms. The CGG repeats are classified into four grades by the American College of Medical Genetics and Genomics, ACMG, according to clinical symptoms and risk of disease onset: 1, 6-44 is referred to as the uninvolved type; 2, 45-54 is called an intermediate type or an ash area; 3, 55-200 is called a pre-mutant; 4, greater than 200, are referred to as full mutants. When the number of CGG repeats is amplified to more than 200, the CGG sequence at the 5' end of the FMR1 gene and the CpG island upstream of the gene are hypermethylated and modified, so that the gene transcription is silenced. FMRP (protein Length polymorphism) encoded by FMR1 is a RNA binding protein, is used as a general regulatory factor in neuronal cells and plays an important role in the formation of nerve synapses. Thus, the whole mutator clinically exhibits severe intellectual impairment and developmental retardation, often accompanied by a predisposition to autism, also known as Fragile X Syndrome (FXS).

The carriers of the premutation of FMR1 gene generally do not have severe intellectual impairment manifestations, but because this gene plays a key regulatory role in RNA transcription and protein translation, the premutation FMR1 gene is thought to be associated with a range of Fragile X-related clinical symptoms, including Fragile X syndrome, Fragile X-related primary ovarian insufficiency (FXPOI), Fragile X-related ataxia (FXTAS).

Tan H2009 in Neuroscientific reports suggests that a CGG repetitive sequence exists in the 5' non-coding region of FMR1 gene, and the length of the CGG repetitive sequence is related to specific clinical symptoms. As shown in FIG. 1, when the number of CGG repeats is 55 or less (left side of FIG. 1), the gene can be normally expressed; when the number of CGG repeats is within the pre-mutation interval (middle part of FIG. 1), a decrease in the amount of protein expression causes a surge in the amount of transcription, and high levels of mRNA lead to FXTAS/FXPOI; when the number of CGG repeats is greater than 200, hypermethylation triggers gene silencing and the patient exhibits severe intellectual impairment, i.e. fragile X syndrome.

In addition, the FMR1 gene of the pre-mutant type showed high instability during passage. In maternal inheritance (maternal inheritance), the CGG sequence of the premutation FMR1 gene has a significant tendency to be amplified, and the number of CGG repeats in offspring may be amplified to 200 or more, leading to the development of a full mutation type (i.e., fragile X syndrome patients). Therefore, there is a need for a method for objectively and accurately assessing the fertility risk of female carriers of premutation.

Current studies indicate that the amplification pattern of CGG sequences is mainly determined by three factors: carrier gender, CGG length, and number of AGGs. CGG sequences from male carriers generally do not show substantial amplification; in female carriers, the length of the CGG sequence is proportional to the risk of amplification to the full mutation, the shortest sequence observed to amplify to the full mutation within one generation is 56; in addition to sex and length, the number of AGG in the CGG sequence plays an important role in stabilizing the CGG sequence. In the CGG sequence of the general population, AGG appears every 9-11 CGG from the 5' end, and the CGG sequence is cut off. The number of AGG in the wild type FMR1 gene is typically 2-3, while the number of AGG in the previously mutated CGG sequence is typically 0-2. The deletion of AGG is considered to be an important reason for the dynamic amplification of CGG sequences.

As shown in FIG. 2, the amplification probability greatly increases when the number of CGG repeats is in the 50-80 region. When the number of AGGs is included in the function, the amplification curve appears to shift to the right significantly as the number of AGGs increases, and the probability estimates are more accurate than the right plot. It was confirmed by studies that CGG repeated at 50-59 segments showed 42% instability in total and 96% in the class without AGG; in the classification with one AGG, 51% showed instability; whereas in the two AGG classifications only 5% showed instability. The stability of the product is improved by 19 times.

However, accurate positioning of the AGG is difficult. Current three primer PCR (TP-PCR) can use the "gap" in the capillary peak to indicate the presence of AGG, but cannot locate the relationship between AGG and either FMR1 allele. Since there are two X chromosomes in women, AGG cannot be mapped to a specific chromosome. As shown in FIG. 3, the three-primer amplification method (TP-PCR) can identify the number of AGG but cannot determine which chromosome is located on.

Disclosure of Invention

In view of the above technical problems in the related art, the present invention provides a kit for determining the number and position of AGG insertions in CGG repeat sequence of FMR1 gene and a method for using the same, which can overcome the above disadvantages of the prior art.

In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:

a kit for determining AGG insertion quantity and position in CGG repetitive sequence of FMR1 gene comprises a primer group, PCR reaction liquid, high-efficiency DNA polymerase aiming at the CGG sequence, FMR1 gene pre-mutation control (quality control), FMR1 gene normal control (quality control), ROX marker (specific DNA molecular fragment suitable for capillary electrophoresis) and deionized water;

the primer group comprises an upstream primer F and a downstream primer R which are used for specifically annealing at AGG and amplifying CGG repetitive region of part of FMR1 gene, the sequence of the upstream primer F is CGGCGGCGGCGGCGAA and is shown as SEQ ID NO. 1, the sequence of the downstream primer R is ACCAGCTCCTCCATCTTCTCTTCAG and is shown as SEQ ID NO. 2, and the 5' end of the downstream primer R is marked with a fluorescent probe.

The upstream primer is matched with a CGG repetitive sequence of a non-coding region at the 5 ' end of the FMR1 gene, is complementary matched with an AGG primer at the position of the last one or two bases at the 3 ' end, and is complementary matched with a sequence at the 3 ' end of the non-coding region of the FMR1 gene.

Further, the ROX label (a specific DNA molecule fragment suitable for capillary electrophoresis) carries a specific fluorophore and enables accurate analysis of CGG fragment size, consisting of 20 DNA fragments of different sizes: less than 100bp, 1; 4 pieces of 100bp-200 bp; 201bp-300bp, 2 strips; 3 pieces of 301bp-400 bp; 401bp-500bp, 2 strips; 501bp-600bp, 3 strips; 601bp-700bp, 1 strip; 701bp-800bp, 2 strips; 901bp-1100bp, 2 pieces.

Further, the GC content of all sequences (DNA molecule fragments) in the ROX marker was above 80%.

Further, the PCR reaction solution includes: tris hydrochloric acid, DMSO, betaine or analogue thereof, GC-melt, magnesium chloride, UNG enzyme and dNTPs with specific proportion.

Further, the FMR1 gene pre-mutation control comprises a high GC content DNA sequence equal to 100 CGG repeats in length; the FMR1 gene normal control comprises a high GC content DNA sequence equal to 30 CGG repeats in length.

Further, the deionized water is sterilized at high temperature.

According to another aspect of the present invention, there is provided a method for using the kit for determining the number and position of AGG insertions in CGG repeats of the FMR1 gene, comprising the steps of:

(1) extracting human cell genome DNA, ultraviolet light-splitting and quantifying, OD₂₆₀nm/OD₂₈₀The ratio of nm should be between 1.8-2.0; OD₂₆₀nm/OD₂₃₀The ratio of nm should be greater than 2.0, using deionized water to dilute the genomic DNA concentration to 15-50 ng/. mu.L, then placing 2. mu.L in each PCR tube;

(2) adding 2 μ L of FMR1 gene normal control and FMR1 gene pre-mutation control into parallel PCR tubes,

(3) and (3) PCR amplification: mixing a detected DNA sample, an FMR1 gene full-mutation quality control product, an FMR1 gene normal quality control product, the primer group, DNA polymerase and PCR reaction liquid to form a PCR reaction system, wherein the final concentration of the primers in the PCR reaction is 750nM, the total reaction system is 15 mu L, and the reaction system comprises the following components:

11.45. mu.L of PCR buffer solution,

the primer is 3.0 mu L in volume,

0.05. mu.L of DNA polymerase,

sample DNA, FMR1 pre-mutation control or FMR1 gene normal control, 1.0 μ L;

the PCR reaction can be carried out on a conventional PCR instrument, and the amplification procedure is as follows:

circulating for 1 time at 98 ℃ for 5 minutes;

circulating for 30 times at 97 ℃, 35 seconds, 60 ℃, 35 seconds, 68 ℃ and 4 minutes;

circulating for 1 time at 72 ℃ for 10 minutes;

at 4 ℃, keeping constant, and circulating for 1 time;

(4) fragment analysis: the PCR products were mixed with ROX marker, HiDi formamide in the following proportions and analyzed using a capillary electrophoresis apparatus: Hi-Di formamide 10 μ L, ROX labeled 1 μ L, PCR product, 3 μ L;

(5) according to the fragment analysis result of the gene analyzer, selecting the Peak with the highest signal value as an analysis object, and automatically recording the molecular size of the analysis object by the system, wherein the mark is Peak_iTo Peak_iSubstituting into the following formula to calculate to obtain CGG_iValue of (a), theoretically c₀The value of 83.2, m₀The value of the carbon dioxide is 2.92,

in the formula, CGG_iThe number of repeats of the CGG sequence following AGG is determined because AGG is generally present at positions 9-10 of the 5' end of the CGG sequence (CGG)_i+10) is greater than the shorter total length of the CGG, the AGG is located in the longer pre-mutant sequence, and vice versa in the shorter wild-type CGG sequence.

The invention has the beneficial effects that:

(1) the kit for determining the number and position of AGG inserted in the CGG repetitive sequence of the FMR1 gene can effectively amplify the CGG repetitive sequence of a larger fragment through specific primer group design and PCR (polymerase chain reaction) procedures, clearly and accurately position AGG on a longer or shorter CGG sequence, and realize more accurate analysis on the sequence structure of the FMR1 gene promoter;

(2) the kit for determining the AGG insertion quantity and position in the CGG repetitive sequence of the FMR1 gene can help clinicians to better evaluate the birth risk of female FMR1 gene pre-mutation carriers, avoid unnecessary prenatal diagnosis risks and prevent unnecessary high-risk pregnancies, and can be used for FMR1 gene screening, prenatal diagnosis and reduce the incidence of fragile X syndrome.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 is a schematic representation of the correlation of the CGG repeat length of the FMR1 gene with specific clinical symptoms;

fig. 2 is a schematic representation of the CGG repeat number and probability of amplification to full mutation for the FMR1 gene, left panel (a): ● represents 0 AGG, t.tag represents 1 AGG, ■ represents 2 AGG; right panel (b) is a plot of CGG length and probability of amplification to full mutation as a function, without regard to AGG number;

FIG. 3 is a schematic diagram of the identification of the number of AGGs by the three-primer amplification method (TP-PCR);

FIG. 4 is a graph showing the analysis of the detection results of the sample described in example 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

Example 1

A kit for determining AGG insertion quantity and positions in CGG repetitive sequences of FMR1 genes comprises a primer group, PCR reaction liquid, high-efficiency DNA polymerase aiming at the CGG sequences, FMR1 gene pre-mutation control products (quality control products), FMR1 gene normal type control products (quality control products), ROX markers (specific DNA molecular fragments suitable for capillary electrophoresis) and deionized water.

The primer group comprises an upstream primer F and a downstream primer R which are used for specifically annealing at AGG and amplifying CGG repetitive region of part of FMR1 gene, the sequence of the upstream primer F is CGGCGGCGGCGGCGAA and is shown as SEQ ID NO. 1, the sequence of the downstream primer R is ACCAGCTCCTCCATCTTCTCTTCAG and is shown as SEQ ID NO. 2, and the 5' end of the downstream primer R is marked with a fluorescent probe.

The upstream primer is matched with a CGG repetitive sequence of a non-coding region at the 5 ' end of the FMR1 gene, is complementary matched with an AGG primer at the position of the last one or two bases at the 3 ' end, and is complementary matched with a sequence at the 3 ' end of the non-coding region of the FMR1 gene.

The ROX marker (specific DNA molecule fragment suitable for capillary electrophoresis) carries a specific fluorescent group and can accurately analyze the size of the CGG fragment, and the ROX marker is composed of 20 DNA fragments with different sizes: less than 100bp, 1; 4 pieces of 100bp-200 bp; 201bp-300bp, 2 strips; 3 pieces of 301bp-400 bp; 401bp-500bp, 2 strips; 501bp-600bp, 3 strips; 601bp-700bp, 1 strip; 701bp-800bp, 2 strips; 901bp-1100bp, 2 pieces.

The GC content of all sequences (DNA molecule fragments) in the ROX marker is more than 80%.

The PCR reaction solution comprises: Tris-HCl, DMSO, betaine or its analogues, GC-melt, magnesium chloride, UNG enzyme and dNTPs.

The FMR1 pre-mutation control comprises a high GC content DNA sequence equal to 100 CGG repeats in length; the FMR1 gene normal control comprises a high GC content DNA sequence equal to 30 CGG repeats in length.

The deionized water is sterilized at high temperature.

The use method of the kit for determining the number and position of AGG insertions in CGG repeat sequence of FMR1 gene comprises the following steps:

(1) extracting human cell genome DNA, ultraviolet light-splitting and quantifying, OD₂₆₀nm/OD₂₈₀The ratio of nm should be between 1.8-2.0; OD₂₆₀nm/OD₂₃₀The ratio of nm should be greater than 2.0, using deionized water to dilute the genomic DNA concentration to 15-50 ng/. mu.L, then placing 2. mu.L in each PCR tube;

(2) adding 2 μ L of FMR1 gene normal control and FMR1 gene pre-mutation control into parallel PCR tubes,

(3) and (3) PCR amplification: and mixing the detected DNA sample, the FMR1 gene full mutation quality control product, the FMR1 gene normal quality control product, the primer group, DNA polymerase and PCR reaction liquid to form a PCR reaction system, wherein the final concentration of the primers in the PCR reaction is 750 nM. The total reaction system is 15. mu.L, and the composition of the reaction system is shown in Table 1:

TABLE 1 PCR reaction System

PCR reaction system	Total volume 15. mu.L
		PCR buffer solution	11.45μL
Primer and method for producing the same	3.0μL
		DNA polymerase	0.05μL
Sample DNA (15-50 ng/. mu.L) or quality control product	1.0μL

The PCR reaction was carried out on a conventional PCR instrument, and the amplification procedure is shown in Table 2:

TABLE 2 PCR reaction procedure

(4) Fragment analysis: the PCR product was mixed with ROX-labeled and HiDi-formamide in the following proportions and analyzed using a capillary electrophoresis apparatus. The system is shown in table 3:

TABLE 3 fragment analysis System

Name of the component	Addition amount (μ L)
		Hi-Di formamide	10
ROX marker	1
		PCR product	3
In total	14

(5) According to the fragment analysis result of the gene analyzer, selecting the Peak with the highest signal value as an analysis object, and automatically recording the molecular size of the analysis object by the system, wherein the mark is Peak_i. Will Peak_iSubstituting into the following formula to calculate to obtain CGG_iThe value of (c). Theoretically, c₀The value of 83.2, m₀The value is 2.92.

In the formula, CGG_iI.e., the number of repeats of the CGG sequence following AGG, since AGG is generally present at positions 9-10 of the 5' end of the CGG sequence (CGG)_i+10) is greater than the shorter total length of the CGG, the AGG is located in the longer pre-mutant sequence, and vice versa in the shorter wild-type CGG sequence.

Example 2

As shown in FIG. 4, the test was carried out using the kit described in example 1, and the sample was obtained from a carrier of the FMR1 gene pre-mutant type, in which the number of CGG repeats was 56/30. Substitution of 153.41 into the equation equals 24.04. This number is equal to the number of CGG repeats after AGG on the longer CGG sequence. The first AGG is known to occur after at least 9 CGGs, so it can be seen that the amplification product at 153.41 is derived from the pre-mutant allele (56 CGGs). The higher peak at the front end, from the common AGG insertion in both alleles of 56 CGGs and 30 CGGs, therefore the signal intensity was approximately twice that of the latter. It can be seen that the two FMR1 genes of this female carrier share three AGG insertions, two of which are located in the pre-mutant allele and one of which is located in the wild-type allele.

In conclusion, by means of the technical scheme, the CGG repetitive sequence of a larger fragment can be effectively amplified through specific primer group design and a PCR program, AGG is clearly and accurately positioned on a longer or shorter CGG sequence, and the sequence structure of the FMR1 gene promoter is more accurately analyzed; the kit can help a clinician to better evaluate the birth risk of female FMR1 gene pre-mutation carriers, avoid unnecessary prenatal diagnosis risk, prevent unnecessary high-risk pregnancy, can be used for FMR1 gene screening and prenatal diagnosis, and reduces the incidence of fragile X syndrome.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Beijing Huaruikang Yuan Biotechnology development Co., Ltd

<120> a kit for determining the number and location of AGG insertions in CGG repeat of FMR1 gene and method of use thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cggcggcggc ggcgaa 16

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

accagctcct ccatcttctc ttcag 25