CN114480624A - Stargardt disease mutation detection kit - Google Patents

Abstract

The invention relates to the technical field of biological genes, in particular to a Stargardt disease mutation detection kit, which comprises an amplification primer, a specific probe, a padlock probe, a rolling circle primer, Taq DNA ligase, Bst DNA polymerase, reaction buffer solution and the like, wherein the inventor firstly discovers a new mutation site from G to A of c.215 on an ABCA4 gene and then develops a corresponding kit; the kit comprises the steps of firstly connecting a padlock probe into a circular product under the action of Taq DNA ligase by taking a human DNA fragment as a template, then carrying out isothermal rolling circle amplification under the action of a rolling circle primer, and detecting whether a new mutation site from G to A of c.215 on ABCA4 gene exists in human DNA through agarose gel electrophoresis. The ABCA4 gene mutation spectrum is increased by the new mutation discovered firstly, and meanwhile, the provided kit is strong in specificity, high in sensitivity, free of temperature circulation and simple to operate, and provides guidance for detecting the Stargardt disease clinically.

Description

Stargardt disease mutation detection kit

Technical Field

The invention relates to the technical field of genes, in particular to a kit for detecting mutation of Stargardt disease.

Background

Stargardt disease (STGD1 OMIM #248200), also known as Stargardt macular dystrophy, juvenile macular degeneration or macular fundus, is one of the most common causes of inherited retinal diseases, accounting for 12% of blindness associated with inherited retinal diseases. Stargardt disease was first described by Karl Stargardt in 1909 and clinical manifestations characterized by adolescent macular dystrophy with central vision loss and atrophic changes in the bilateral macular regions, including macular red copper-like changes, bovine eye maculopathy, progressive macular atrophy and yellowish white spots in the posterior pole of the retina; histologically, the disease is closely related to photoreceptor cell death and retinal degeneration caused by the deposition of large amounts of lipofuscin in the retinal pigment epithelium. In 1998, Blacharski et al estimated the global prevalence of Stargardt disease to be 1/10000, while recent reports from the United kingdom eye monitoring office indicated that the annual incidence in the United kingdom was estimated to be 0.110-0.128 per 100,000.

The Stargardt disease inheritance pattern is mainly autosomal recessive inheritance and is caused by a biallelic mutation of the ABCA4 gene. The ABCA4 gene maps to chromosome 1p22.1, consists of 50 exons, and encodes a retinal-specific membrane-associated ABCA4 protein. The ABCA4 protein is a 2273 amino acid protein with a molecular weight of approximately 250kDa, belongs to a member of the ATP-binding cassette transporter superfamily, localizes to the disc membrane border of rod and cone photoreceptor outer segments, and plays an important role in the retinol cycle in the visual cycle. Structurally, the ABCA4 protein is composed of two non-identical but homologous portions, each of which comprises a transmembrane domain, a nucleotide binding domain and a cytoplasmic domain. Mutations in the ABCA4 gene result in dysfunction of the ABCA4 transporter, accumulation of toxic tretinoin, and degeneration of the retinal pigment epithelium and photoreceptors, resulting in irreversible central vision loss.

The ABCA4 gene is first discovered by Allikmets et al in 1997, so far, more than 2000 ABCA4 mutations (www.lovd.nl/ABCA4) have been reported in the literature, but many patients still cannot obtain clear diagnosis, the pathogenic site related to Stargardt disease still needs to be further researched, and the discovery of any new mutation has an important role in the diagnosis and prevention of the disease.

Therefore, a rapid, simple, accurate and economical gene mutation detection method is developed, and has important significance for screening ABCA4 gene mutation of Stargardt patients.

Disclosure of Invention

Based on the reasons, the invention provides a Stargardt disease mutation detection kit, which comprises an amplification primer, a specific probe, a padlock probe, a rolling circle primer, Taq DNA ligase, Bst DNA polymerase, reaction buffer solution and the like; the kit comprises the steps of firstly connecting a padlock probe into a circular product under the action of TaqDNA ligase by taking a human DNA fragment as a template, then carrying out isothermal rolling circle amplification under the action of a rolling circle primer, and detecting whether a new mutation site from G to A of c.215 on ABCA4 gene exists in human DNA through agarose gel electrophoresis. The ABCA4 gene mutation spectrum is increased by the new mutation discovered firstly, and meanwhile, the provided kit is strong in specificity, high in sensitivity, free of temperature circulation and simple to operate, and provides guidance for detecting the Stargardt disease clinically.

According to the invention, a family with Stargardt disease in China is firstly researched, a new mutation site on ABCA4 gene, specifically a mutation site from G to A of c.215, is firstly discovered through whole exon sequencing, and family members and 200 normal controls are verified and subjected to cosegregation analysis (shown in figure 2) through Sanger sequencing, so that the family members are finally determined to be the new mutation site. The corresponding inventors disclose that the DNA fragment with the mutation site has the nucleotide sequence shown as Seq ID No. 1, the coded amino acid sequence shown as Seq ID No. 2, the non-mutated DNA fragment has the nucleotide sequence shown as Seq ID No. 3, and the coded amino acid sequence shown as Seq ID No. 4, and according to the comparison, the mutated DNA fragment contains the c.215G > A mutation, and the corresponding amino acid sequence has the p.G72E mutation.

On the basis of the above findings, the inventors further provide a set of detection primers:

the upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', the nucleotide sequence of the upstream primer is shown as Seq ID No. 5,

the downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', the nucleotide sequence of which is shown as Seq ID No. 6, and can be used for detecting the mutation and amplifying the DNA fragment;

further, the inventors provide a kit for detecting the above mutation, which mainly consists of:

10 mu M amplification primer, 10 mu M specific probe, 10 mu M padlock probe, 10 mu M rolling circle primer, Taq DNA ligase, Bst DNA polymerase and reaction buffer solution; the more specific composition is as follows:

an amplification primer, wherein the upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', the nucleotide sequence of the amplification primer is shown as Seq ID No. 5, the downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', and the nucleotide sequence of the amplification primer is shown as Seq ID No. 6;

specific probes (control probes) include:

mutant probe 5'-GGCTCCAGGAGATCTTCTGC-3' having the nucleotide sequence shown in Seq ID No. 7 (as a positive control);

wild-type probe 5'-GGCTCCAGGGGATCTTCTGC-3', whose nucleotide sequence is shown in Seq ID No:8 (as a negative control);

padlock probe 5-CCTGGAGCCAAATACAGATGAATGTGTCCTACTCGAGTCCTTAAC CAAAATGTATAAAGCAGAAGATCT-3', the nucleotide sequence of which is shown in Seq ID No. 9;

a rolling circle primer 5'-GTTAAGGACTCGAGTAGGA-3', the nucleotide sequence of which is shown as Seq ID No. 10;

in addition, the kit also contains

40U/. mu.l Taq DNA ligase; 8U/. mu.l Taq Bst DNA polymerase; the reaction buffer comprises 2 XTaq PCR pre-mixed solution, 1 XTaq DNA ligase reaction buffer, 1 XBst DNA polymerase reaction buffer and 1mM dNTPs.

The corresponding reaction system of the kit is as follows:

control group: (Positive control and negative control in each group)

The ligation reaction was a 10. mu.l system comprising 2.5. mu.l of 10. mu.M specific probe (negative or positive control), 1. mu.l of 10. mu.M padlock probe, 0.1. mu.l of 40U/. mu.l Taq DNA ligase, 1. mu.l Taq DNA ligase reaction buffer and 5.4. mu.l ribozyme-free water;

the polymerization reaction was a 10. mu.l system comprising 7. mu.l of the ligation product, 0.8. mu.l of 1mM dNTPs, 0.7. mu.l of 8U/. mu.l Bst DNA polymerase, 1. mu.l of Bst DNA polymerase reaction buffer and 0.5. mu.l of ribozyme-free water;

the specific probe adopted by the control group can serve as a template and a primer, so that a rolling circle primer does not need to be additionally added.

And (3) sample group to be tested:

the ligation reaction was a 10. mu.l system comprising 2.5. mu.l of human DNA sample (concentration 60 ng/. mu.l), 1. mu.l of 10. mu.M padlock probe, 0.1. mu.l of 40U/. mu.l Taq DNA ligase, 1. mu.l Taq DNA ligase reaction buffer and 5.4. mu.l ribozyme-free water;

the polymerization reaction was a 10. mu.l system comprising 7. mu.l ligation product, 0.3. mu.l 10. mu.M rolling circle primer, 0.8. mu.l 1mM dNTPs, 0.7. mu.l 8U/. mu.l Bst DNA polymerase, 1. mu.l Bst DNA polymerase reaction buffer and 0.2. mu.l ribozyme-free water.

The specific use method of the kit is as follows:

(1) sample pretreatment:

extraction of genomic DNA: extracting genome DNA from a sample to be detected by utilizing the prior art;

amplification of the fragment of interest: carrying out PCR amplification by using amplification primers (shown in Seq ID No:5 and 6) provided by the kit, wherein the size of an amplification product is 79 bp;

and (3) purification of an amplification product: purifying a DNA fragment to be detected from an amplification product by using the prior art;

(2) pre-denaturation: mixing 2.5 μ l human DNA sample (or specific probe) and 1 μ l padlock probe, pre-denaturing at 95 deg.C for 10min, and slowly reducing to 40 deg.C at 0.02 deg.C/s;

(3) connecting: the system is the above-mentioned 10 mul connection system, the reaction condition is 68 deg.C, 30 min;

(4) polymerization: the system is the 10 mul polymerization system, the reaction condition is 56 ℃, 2.5 h;

(5) electrophoresis: and (4) taking the polymerized rolling circle amplification product to perform agarose gel electrophoresis and analyzing.

The result judgment criteria are as follows:

in an agarose electrophoresis picture, for a mutant, because the mutation exists, rolling circle amplification occurs, the molecular weight is large, electrophoresis cannot be carried out, and a band is retained at a sample loading hole; the wild type does not generate rolling circle amplification, the molecular weight is small, and no band exists at the sample loading hole.

In conclusion, the kit provided by the invention can detect the mutation from the Stargardt disease related gene ABCA4 c.215G to A in a short time by combining with a rolling circle amplification technology, enriches the diagnosis sites of the Stargardt disease diagnosis kit, has the advantages of strong specificity, high accuracy, high sensitivity, no need of temperature circulation and simple operation, provides guidance for clinical detection of the Stargardt disease, and further has very important significance for screening and preventing the Stargardt disease.

Drawings

FIG. 1 is a pedigree profile of Stargardt patients with corresponding mutations according to the invention,

FIG. 2 is a comparison of the Sanger sequencing verification peaks of the pedigree of FIG. 1 with the presence or absence of mutation in the ABCA4 gene c.215G > A,

wherein I-1 represents the Sanger sequencing peak plot of one member with the mutation in the family plot, referring to member No. 1 at generation I; i-2 represents the Sanger sequencing peak plot of one member without the mutation in the family plot, referring to member I and member No. 2;

FIG. 3 is a schematic diagram of the ligation principle of the Taq DNA ligase action ABCA4 mutant gene c.215G > A provided by the invention,

the mutant sequence (MT) can be completely complementary and paired with the padlock probe (P) to form a cyclic product under the action of ligase, and the wild-type sequence (WT) is mismatched with the padlock probe (P) and cannot be connected into the cyclic product;

FIG. 4 is a schematic diagram of the amplification of a control group provided by the present invention,

connecting the padlock probes by taking the mutant probes as templates to form a ring product under the action of TaqDNA ligase, and performing rolling ring amplification by taking the mutant probes as primers under the action of Bst polymerase to amplify the rolling ring product; the wild probe and the padlock probe form mismatch, and the padlock probe cannot be connected into a circular product, so that rolling circle amplification cannot be carried out;

FIG. 5 is a diagram of agarose gel electrophoresis for detecting mutation in a control group according to the present invention,

lanes 1 and 2 are the reaction products to which only the specific probe and padlock probe are added, and lanes 3 and 4 are the reaction products to which the specific probe, padlock probe and Taq DNA ligase are added; lanes 5 and 6 are the reaction product with ligation product and dNTP, lanes 7 and 8 are the product with ligation product, dNTP and Bst polymerase, where lanes 7 and 8 are the positive and negative control results of the present invention, and it can be seen that there is a band at the upper well of lane 7;

FIG. 6 is a schematic diagram of the amplification of a sample set to be tested according to the present invention,

firstly, extracting DNA (gDNA) in a sample to be detected, amplifying a 79bp fragment (dsDNA) by PCR (polymerase chain reaction), then denaturing the dsDNA into a single-stranded DNA (ssDNA), connecting the sample to be detected containing mutation by using a padlock probe under the action of Taq DNA ligase and using a mutant DNA chain (MT-ssDNA) as a template to form a ring product, and performing rolling ring amplification in the presence of a rolling ring primer and Bst polymerase to amplify the rolling ring product; the sample to be detected which does not contain mutation, namely a wild type DNA chain (WT-ssDNA) and the padlock probe form mismatch, and the padlock probe cannot be connected into a circular product, so that rolling circle amplification cannot be carried out;

FIG. 7 is a diagram of the agarose gel electrophoresis for detecting mutation in a sample set to be tested according to the invention,

lanes 2 and 3 show the electrophoresis of the ligation products of members I-1 and I-2 in the family of FIG. 1, and lanes 4 and 5 show the electrophoresis of the products of the polymerization of both, showing that the mutation is present in the member I-1 (lane 4) and not in the member I-2 (lane 5), which is consistent with the Sanger sequencing in FIG. 2.

Detailed Description

In the context of this specification, unless otherwise indicated, any terms used herein have the meanings commonly understood in the art by those skilled in the art, and experimental procedures without specification to details are carried out according to conventional experimental procedures or according to the instructions recommended by the supplier.

Example 1 mutation site determination

Firstly, a family (shown in figure 1) suffering from Stargardt disease in China is researched, a new mutation site on ABCA4 gene, specifically a mutation site from G to A of c.215, is found for the first time through whole exon sequencing, and family members and 200 normal controls are verified and subjected to cosegregation analysis (shown in figure 2) through Sanger sequencing, and finally the family members and 200 normal controls are determined to be the new mutation site. The corresponding inventors disclose that the DNA fragment with the mutation site has the nucleotide sequence shown as Seq ID No. 1, the coded amino acid sequence shown as Seq ID No. 2, the non-mutated DNA fragment has the nucleotide sequence shown as Seq ID No. 3, and the coded amino acid sequence shown as Seq ID No. 4, and according to the comparison, the mutated DNA fragment contains the c.215G > A mutation, and the corresponding amino acid sequence has the p.G72E mutation.

EXAMPLE 2 composition of the assay kit

The inventors provide a set of detection primers:

the upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', the nucleotide sequence of the upstream primer is shown as Seq ID No. 5,

the downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', the nucleotide sequence of which is shown as Seq ID No. 6, and can be used for the amplification of the DNA fragment;

further, the inventors provide a kit for detecting the above mutation, which mainly consists of: 10 mu M amplification primer, 10 mu M specific probe, 10 mu M padlock probe, 10 mu M rolling circle primer, Taq DNA ligase, Bst DNA polymerase and reaction buffer solution; the more specific composition is as follows:

an amplification primer, wherein the upstream primer is 5'-CACTACACCGGGAGCTTGAGA-3', the nucleotide sequence of the amplification primer is shown as Seq ID No. 5, the downstream primer is 5'-TGACCAGAAGGCAGTGGACACAT-3', and the nucleotide sequence of the amplification primer is shown as Seq ID No. 6;

specific primer probes (control probes) include:

mutant primer probe 5'-GGCTCCAGGAGATCTTCTGC-3' having the nucleotide sequence shown in Seq ID No. 7 (as a positive control);

wild-type primer probe 5'-GGCTCCAGGGGATCTTCTGC-3', whose nucleotide sequence is shown in Seq ID No:8 (as negative control);

lock type probe 5'-CCTGGAGCCAAATACAGATGAATGTGTCCTACTCGAGTCCTTAAC CAAAATGTATAAAGCAGAAGATCT-3', the nucleotide sequence of which is shown in Seq ID No. 9;

a rolling circle primer 5'-GTTAAGGACTCGAGTAGGA-3', the nucleotide sequence of which is shown as Seq ID No. 10;

in addition, the kit also contains

40U/. mu.l Taq DNA ligase; 8U/. mu.l Taq Bst DNA polymerase; the reaction buffer comprises 2 XTaq PCR pre-mixed solution, 1 XTaq DNA ligase reaction buffer, 1 XBst DNA polymerase reaction buffer and 1mM dNTPs.

The corresponding reaction system of the kit is as follows:

control group: (Positive control and negative control in each group)

The ligation reaction was a 10. mu.l system comprising 2.5. mu.l of 10. mu.M specific probe (negative or positive control), 1. mu.l of 10. mu.M padlock probe, 0.1. mu.l of 40U/. mu.l Taq DNA ligase, 1. mu.l Taq DNA ligase reaction buffer and 5.4. mu.l ribozyme-free water;

the polymerization reaction was a 10. mu.l system comprising 7. mu.l of the ligation product, 0.8. mu.l of 1mM dNTPs, 0.7. mu.l of 8U/. mu.l Bst DNA polymerase, 1. mu.l of Bst DNA polymerase reaction buffer and 0.5. mu.l of ribozyme-free water;

the specific probe adopted by the control group can serve as a template and a primer, so that a rolling circle primer is not required to be additionally added.

And (3) sample group to be tested:

the ligation reaction was a 10. mu.l system comprising 2.5. mu.l of human DNA sample (concentration 60 ng/. mu.l), 1. mu.l of 10. mu.M padlock probe, 0.1. mu.l of 40U/. mu.l Taq DNA ligase, 1. mu.l Taq DNA ligase reaction buffer and 5.4. mu.l ribozyme-free water;

the polymerization reaction was a 10. mu.l system comprising 7. mu.l ligation product, 0.3. mu.l 10. mu.M rolling circle primer, 0.8. mu.l 1mM dNTPs, 0.7. mu.l 8U/. mu.l Bst DNA polymerase, 1. mu.l Bst DNA polymerase reaction buffer and 0.2. mu.l ribozyme-free water.

EXAMPLE 3 use of the kit

The method for detecting the mutation of ABCA4 gene c.215G > A by using the kit prepared in example 2 comprises the following specific steps:

step 1, sample pretreatment

(1) The extraction of genomic DNA was carried out using a conventional DNA extraction kit (in this example, Axygen manufactured by Corning Corp., product number AP-MD-FBL-GDNA-20), and the specific steps were as follows:

1) adding 2ml of blood sample to be tested into 5ml of Buffer AP1, and violently mixing uniformly;

2) adding 1ml Buffer AP2, mixing immediately, centrifuging 4630g for 15 min;

3) transferring the supernatant into a medium preparation tube, starting negative pressure, maintaining the negative pressure, and adding 7ml of Buffer W1, 8ml of Buffer W2 and 4ml of Buffer W2;

4) discharging 12000g of the tube head of the middle burette, and centrifuging for 2 min;

5) adding 0.4ml of Elution buffer, standing at room temperature for 5min, centrifuging at 12000g for 2min, and storing at-20 ℃ for later use.

(2) Amplification of the fragment of interest: PCR amplification is carried out by using the amplification primer provided by the kit to obtain a DNA fragment with the size of 79 bp.

PCR amplification was performed according to the following system and procedure:

and (3) PCR reaction system:

PCR reaction procedure:

(3) the PCR product was purified using a conventional PCR product purification kit (in this example, SanPrep column PCR product purification kit available from Shanghai, cat. B518141-0050), and the specific steps were as follows:

1) adding Buffer B3 with the volume of 5 times of that of the PCR reaction solution, and fully and uniformly mixing;

2) centrifuging at 8000g for 30 s, and pouring out liquid in the collecting pipe;

3) adding 500 mul Wash Solution, centrifuging at 9000g for 30 seconds, and pouring out the liquid in the collecting pipe;

4) repeating the step 3) once;

5) the empty column 9000g was centrifuged for 1 min;

6) the adsorption column was placed in a clean 1.5ml centrifuge tube, 20. mu.l of Elution Buffer was added to the center of the adsorption membrane, and after standing at room temperature for 1 minute, it was centrifuged for 1 minute and stored at-20 ℃.

Step 2, Pre-denaturation

Mu.l of human DNA sample (or specific probe) and 1. mu.l of padlock probe were mixed and pre-denatured under 95 ℃ for 10min, and then slowly reduced to 40 ℃ at a rate of 0.02 ℃/s.

Step 3, connecting

The ligation reaction was a 10. mu.l system comprising 2.5. mu.l of 10. mu.M human sample DNA (60 ng/. mu.l) or 10. mu.M specific probes (negative and positive controls), 1. mu.l of 10. mu.M padlock probe, 0.1. mu.l of 40U/. mu.l Taq DNA ligase, 1. mu.l Taq DNA ligase reaction buffer and 5.4. mu.l ribozyme-free water; the reaction conditions were 68 ℃ for 30 min.

Step 4, polymerization

The polymerization reaction was a 10. mu.l system comprising 7. mu.l ligation product, 0.3. mu.l 10. mu.M rolling circle primer, 0.8. mu.l 1mM dNTPs, 0.7. mu.l 8U/. mu.l Bst DNA polymerase, 1. mu.l Bst DNA polymerase reaction buffer and 0.2. mu.l ribozyme-free water (control group did not require the addition of rolling circle primer, 0.5. mu.l ribozyme-free water was added); the reaction conditions were 56 ℃ for 2.5 h.

Step 5, electrophoresis

Taking the polymerized rolling circle amplification product to perform agarose gel electrophoresis and analyzing:

(1) preparing gel (2% agarose), weighing 0.8g agarose and dissolving in 40ml 1xTBE solution;

(2) sol: heating in a microwave oven to boil, taking out, adding 2 mu l G-red, and heating in the microwave oven to boil;

(3) spreading glue: pouring 40ml of glue solution into the glue groove, and inserting a comb ruler;

(4) gluing: putting the glue groove and the glue into an electrophoresis tank containing 1xTAE liquid, wherein the liquid level is 1-2mm away from the glue surface, and pulling out a comb ruler;

(5) sample adding: adding the amplification product mixed with the loading buffer into the glue hole;

(6) glue spreading: covering an electrophoresis tank cover, checking the anode and the cathode, starting an electrophoresis apparatus, and adjusting the voltage to 110V to run glue;

(7) and (3) photographing: when electrophoresis is carried out for 40min, the electrophoresis apparatus is closed, the gel is carefully taken out, and the gel is placed into an imager for photographing.

FIG. 5 is an agarose gel electrophoresis of a control, showing that the mutant products remained in the wells (positive control) and the wild type wells were not banded (negative control), as shown in lanes 7 and 8; FIG. 7 is an electrophoretogram of a test sample set, as shown in lanes 4 and 5, the mutation is present in the family member I-1, and the mutation is absent in the member I-2, consistent with the Sanger sequencing results in FIG. 2. The kit can detect the ABCA4 c.215G to A mutation of the gene related to the Stargardt disease in a short time.

It should be understood that the above embodiments are only for understanding the technical solution and the core principle of the present invention, and are not to be construed as limiting the scope of the present invention. For those skilled in the art, the conditions and parameters of the present embodiment can be changed according to the needs based on the core principle of the present invention, but these equivalent changes and modifications still belong to the protection scope of the present invention.

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Claims (3)

1. A Stargardt disease mutation detection kit is characterized in that: the main components are as follows:

10 mu M amplification primer, 10 mu M padlock probe, 10 mu M specific probe, 10 mu M rolling circle primer, Taq DNA ligase, Bst DNA polymerase and reaction buffer solution;

the amplification primers are as follows: the nucleotide sequence of the upstream primer is shown as Seq ID No. 5, and the nucleotide sequence of the downstream primer is shown as Seq ID No. 6;

wherein the specific probe comprises a mutant probe and a wild primer probe, the nucleotide sequence of the mutant probe is shown as Seq ID No. 7, and the nucleotide sequence of the wild primer probe is shown as Seq ID No. 8;

the nucleotide sequence of the padlock probe is shown as Seq ID No. 9;

the nucleotide sequence of the rolling circle primer is shown as Seq ID No. 10.

2. The Stargardt disease mutation detection kit according to claim 1, wherein: the kit also contains 40U/. mu.l Taq DNA ligase; 8U/. mu.l Taq Bst DNA polymerase; the reaction buffer comprises 2 XTaq PCR pre-mixed solution, 1 XTaq DNA ligase reaction buffer, 1 XBst DNA polymerase reaction buffer and 1mM dNTPs.

3. A section of ABCA4 gene DNA segment with c.215G & gtA mutation point, which is characterized in that: the nucleotide sequence is shown as Seq ID No. 1, and the coded amino acid sequence is shown as Seq ID No. 2.

Images