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Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia

A technology of thalassemia and nucleic acid hybridization, which is applied in the field of nucleic acid hybridization membrane strips for diagnosing thalassemia, and can solve problems such as difficult to distinguish, difficult to judge, and poor signal uniformity

Active Publication Date: 2008-06-25
亚能生物技术(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the mutation probe and the normal probe are at the same position on the glass slide, and are only distinguished by different colors, so that the samples of mutant heterozygotes have mixed colors, which is difficult to judge; Poor monotony, often with false signals, sometimes difficult to distinguish between false signals and mutant heterozygotes when mixed signals occur
[0014] So far, there are no reports of nucleic acid hybridization membrane strips for β-thalassemia based on nylon membranes or with mutant genes in the middle of oligonucleotide probes

Method used

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  • Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia
  • Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia
  • Nucleic acid hybrid film strip and reagent box used for diagnosing beta mediterranean sea thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] [Example 1] Preparation of nucleic acid hybridization membrane strips for detecting β-thalassaemia

[0080] 1. Preparation of probes

[0081] Probes were synthesized according to the sequences in Table 3, and the probes with reverse complementary sequences had the same effect as the sequences in the table, and will not be listed one by one in this table. Carrying out amino labeling on the 5' end or 3' end of the probe has the same effect, and the synthesis method is a conventional DNA synthesis method.

[0082] Table 3 DNA sequence of mutation probe

[0083] serial number

[0084] Note: W=A / T in the sequence of the probe IVS1-1M of SEQ ID NO.9.

[0085] Table 5 DNA sequence of normal probe

[0086] serial number

[0087] 2. Fabrication of nucleic acid hybridization strips

[0088] After the synthesis of oligonucleotide probes is completed, direct oligonucleotide probe immobilization is required. First cut the nylon membrane into a certain size, ...

Embodiment 2

[0092] [Example 2] Design of primers for amplifying the DNA in the sample to be tested

[0093] According to the sequence data of the β-thalassemia gene mutation, two pairs of specific primers with different lengths and Tm values ​​and containing a biotin label at the 5' end were used to amplify two fragments of the β-globin gene. The nucleotide sequences of these two pairs of primers are shown in Table 6. In this table, the amplified products of SEQ ID NO.27 and SEQ ID NO.28 can be hybridized with 654N and / or 654M for detecting the mutation at position 654; the amplification of SEQ ID NO.25 and SEQ ID NO.26 The product was used to detect mutations at other sites except 654.

[0094] Table 6 DNA sequences of β-globin gene PCR primers of different lengths

[0095] serial number

Embodiment 3

[0096] [Example 3] Detection of sample DNA to be tested

[0097] 1. Experimental instruments and reagents

[0098] 1. Test equipment

[0099] PCR instrument, DNA electrophoresis instrument, molecular hybridization box, shaker

[0100] 2. Test reagents

[0101] (1) 3% H 2 o 2

[0102] (2) 20×SSC (pH7.0): take 175.3g of NaCl, 88.2g of sodium citrate, add H 2 Dissolve O to 1000 mL, adjust the pH to 7.0 with a pH meter, and autoclave.

[0103] (3) 10% SDS (pH 7.0): Dissolve 20g of SDS in 180mL of H2O, adjust the pH to 7.0 with HCl, and finally set the volume to 200mL.

[0104] (4) Liquid A (2×SSC, 0.1% SDS, pH 7.4): take 100mL of 20×SSC, 10mL of 10% SDS, add H 2 O was dissolved to 1000mL, and the pH was adjusted to 7.4.

[0105] (5) Solution B (0.5×SSC, 0.1% SDS, pH 7.4): take 20×SSC 25mL, 10% SDS 10mL, add H 2 O was dissolved to 1000mL, and the pH was adjusted to 7.4.

[0106] (6) Liquid C (0.1M sodium citrate, pH 5.4): Dissolve 14.7 g of sodium citrate in 450 mL of wate...

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Abstract

A nucleic acid hybridization membrane strip used for diagnosing the beta-Mediterranean anema is composed of a substrate and the specific probes fixed to the substrate. Said each specific probe (mutant probe) is relative to one of the mutant beta-globin gene sites and has the sequence of 14-20 bases. Its reagent kit is also disclosed.

Description

technical field [0001] The invention relates to a nucleic acid hybridization membrane strip for diagnosing thalassemia, in particular to a nucleic acid hybridization membrane strip for diagnosing beta-thalassemia. [0002] The invention also relates to PCR primers for amplifying DNA in a sample to be tested. [0003] The invention also relates to a kit for diagnosing beta-thalassemia. Background technique [0004] Thalassemia (thalassemia for short) is a common genetic disease that occurs all over the world. It is most common in the Mediterranean region in the west, Turkey, Middle East countries in the middle, Southeast Asia and southern my country in the east. The clinical phenotypes of thalassemia are diverse, ranging from normal to requiring repeated blood transfusions, and even life-threatening, leading to premature death or stillbirth in patients. Due to the high incidence of the disease in the south of my country, it creates enormous mental and economic pressure on s...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 曲敬廖生赟周逸欧阳理
Owner 亚能生物技术(深圳)有限公司
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