CN1062018C - Detecting technique for human haemoglobin (globin) a* gene and gene a* - Google Patents

Abstract

A genetic detection method, which uses DNA polymerase chain reaction (PCR) technology to carry out genotyping detection for the deletion of human hemoglobin (globin) α ₁ gene and α ₂ gene. The present invention designs three pairs of amplification primers αp ₁ and αp ₃ , αp ₁ and αp ₂ , βp ₁ and βp ₂ with a clear arrangement order, which respectively amplify Hbα ₁ gene 278bp, Hbα ₂ gene fragment 603bp, and Hbβ gene fragment 400bp Carry out the amplification reaction with the matching experimental technique of the amplification reaction, i.e. the reagent system of the amplification reaction and its reaction program, and the product is detected by electrophoresis, which can accurately determine the tested human hemoglobin (globin) α ₁ gene and α ₂ gene Number of missing α genes. It is of great significance to scientific research and medical testing. The invention is sensitive, accurate, easy to operate and low in price.

Description

Detection Technology of Human Hemoglobin (Globin) α1 Gene and α2 Gene

The invention belongs to the field of genetic detection, and is a technique for detecting human hemoglobin (globin) _α1 gene and _α2 gene by using a DNA polymerase chain reaction (PCR) method.

Loss of the _α1 or _α2 gene encoding hemoglobin (globin) causes α-thalassemia (thalassemia for short). It is a common disease in my country, with an incidence rate of 2.64%, and it is as high as 14.95% in some areas of Guangxi. At present, there is no effective cure method, and severe patients need blood transfusions all year round. come a heavy burden.

α-thalassemia is mostly caused by the deletion of α-gene except for a few caused by point mutation of α-gene. There are two _α1 and _α1 genes with very similar sequences in the α-gene family. There are only 7 nucleotide sequence differences between the two. They are functionally identical, and both encode α-globin molecules. Deletion or partial deletion of both genes reduces α-globin synthesis. There are 2 _α1 alleles and 2 _α2 alleles in the normal human genome. Generally, patients who lack one α gene manifest as α-thalassemia 2, with mild or no symptoms of anemia; patients who lack 2 α genes exhibit α-thalassemia 1, with severe symptoms of anemia; those who lack 3 α genes The patient showed HbH disease, severe symptoms of anemia, and needed blood transfusion all year round; the fetus lacking 4 α genes showed Bart's edema and could not survive.

At present, red blood cell morphology observation and hemoglobin electrophoresis analysis are used to detect the deletion of human hemoglobin (globin) α ₁ gene and α ₂ gene. Methods such as restriction endonuclease and α gene probe hybridization can also be used to detect the α gene deletion. For research, these methods are either technically complex or difficult to grasp objective standards. Since 1987, a pair of primers designed in the α gene family ψα ₁ (α ₁ pseudogene) gene has been generally used to carry out PCR (Polymerase chain reaction Polymerase chain reaction) for the deletion of human hemoglobin (globin) α ₁ gene or α ₂ gene Reaction) amplification, if a large fragment of the α gene family including α ₁ and α ₂ genes is deleted, the correct detection result can be obtained; if the deletion is only α ₁ and α ₂ genes or α ₁ or α ₂ genes False-negative results were obtained when _ψα1 was not included; false-positive results were obtained if only the _ψα1 gene was deleted and the _α1 and _α2 genes or _α1 or _α2 genes were not deleted. Therefore, this PCR detection method has great limitations. Moreover, most of the α-thalassemia patients in China have _α1 and _α2 genes or _α1 or _α2 gene deletions, and the application of this method may easily lead to detection errors.

The purpose of the present invention is to establish a PCR genotyping detection technology of human hemoglobin (globin) _α1 gene and _α2 gene with simple operation, convenient method and unmistakable detection standard.

The invention uses the PCR amplification technology, mainly including the main steps of primer formation, gene amplification and display detection. Invention designed five nucleotide primers, which are:

αp ₁ : 5′d(CGG CTC TGC CCA GGT TAA GG)

αp ₁ : 5′d(CCT TGG TCT GAG ACA GGT AAA CA)

αp ₁ : 5′d(AGT GGG GCC GAG GGC CCA G)

βp ₁ : 5′d(AAG GAG ACC AAT AGA AAC TGG GC)

βp ₁ : 5′d(TCT CCC CTT CCT ATG ACA TGA AC)

Among them, αp ₁ and αp ₂ primers amplify Hbα ₁ gene fragment 278bP, αp ₁ and αp ₂ amplify Hbα: gene fragment 603bp, βp ₁ and βp ₂ amplify Hbβ gene fragment 400bP. The five primers formed three pairs respectively, and the amplified fragments had obvious characteristics.

The primers αp ₁ , αp ₂ , αp ₃ , βp ₁ , and βp ₂ can be synthesized by chemical methods according to the above nucleotide sequence. 278bp, 603bp, 400bp DNA fragments can be detected by electrophoresis. For example, the DNA fragments are separated by ordinary agarose gel electrophoresis and stained with ethidium bromide, and detected by red fluorescence under ultraviolet light.

The reagent system for gene amplification can contain 10-50mM Tris-HCl (PH=8.1-8.5), 50mM KCl, 0.5-3.0mM NgCl ₂ , 10-25mM (NH ₄ ) ₂ SO ₄ , gelatin 200 μg/ml, 4-8% formazan, dNTP (A·G·C·T) 200-300 μM each, 1-1.5U heat-resistant DNA polymerase, 0.25-0.5 μM of αp ₁ , 0.125-0.25 μM, αp ₁ , 0.125-0.25 μM of αp ₁ , 0.125-0.25 μM of βp ₁ , 0.125-0.25 μM of βp ₁ .

Concentrate the above reagents by 10 times except for the heat-resistant DNA polymerase, and prepare the primers according to the required concentration to form a kit, which is more convenient to use.

Using the above-mentioned PCR reaction reagent system, the electrophoresis bands of the PCR amplification products are clear and bright without miscellaneous bands, and are easy to identify.

During the amplification reaction, first mix the reaction system well, then centrifuge at high speed for 30 seconds, then denature at 90-93°C for 3-10 minutes, add enzyme, beat well, and then centrifuge; at 90-93°C-0.5 -1 minute, 50-60°C for 0.5-1 minute, 68-72°C for 1-2 minutes for 30-35 cycles; then keep warm at 68-72°C for several minutes, take the reaction product on the agarose gel plate Electrophoresis, observe the electrophoresis band under ultraviolet light irradiation. Use Hybaid or FR-300 PCR amplification instrument or constant temperature water bath to amplify, and adjust the temperature and incubation time appropriately within the above range.

Under this amplification program, the amplified product has _clear swimming bands and high sensitivity when electrophoresis _shows detection. The distances between the electrophoresis bands of the three gene fragments are equal and easy to identify. Coupled with the amplification reagent system and amplification program of the present invention, the three electrophoresis bands are clear and bright, and the brightness of the bands is similar.

Referring to the standard of 400 bp of βp ₁ and βp ₂ gene fragments, if the tested human hemoglobin (globin) α gene is heterozygous for the deletion of α ₁ gene or α ₂ gene, the amplification of their α ₁ gene or α ₂ gene fragment The brightness of the product electrophoresis band is only less than half of the brightness of the 400bp band of the β gene amplification product.

If the tested human hemoglobin (globin) α gene is homozygous for the deletion of α ₁ gene and α ₂ gene, there will only be a 400bp band of the amplified product of β gene, but no αp ₁ and αp ₃ , αp ₁ and αp ₂ Amplified 278bp, 603bp electrophoresis bands. If one _α1 gene or _α2 gene is missing in the _α1 gene and _α2 gene of the α gene family with similar sequences, it is a heterozygote, and the lack of two allele α genes is a homozygote.

If the tested human hemoglobin (globin) α gene lacks 3 α genes, the electrophoresis band of the amplified product of the α ₁ gene or α ₂ gene fragment has only one band, and its brightness is less than half of the brightness of the β band;

If the tested human hemoglobin (globin) α gene lacks 4 α genes, there will be no electrophoresis bands of the amplified product of α ₁ gene and α ₂ gene fragments.

Figure 1 is an electropherogram showing detection, 1 is the 278 bp Hbα ₁ gene fragment amplified by αp ₁ and αp ₃ , 2 is the 603 bp Hbα ₂ gene fragment amplified by αp _{1 and} αp 2, and 3 is the amplification of βp ₁ and βp ₂ The Hbβ gene fragment is 400bp. The blackness in the figure indicates the brightness of the electrophoresis band, row 4 is the electrophoresis band of φX174(RF)-HaeⅢ, row 5 is the electrophoresis band without deletion of the hemoglobin (globin) α gene, that is, the normal human hemoglobin (globin) α gene detection The result, line 6 is the electrophoresis band after the amplification of the human hemoglobin (globin) α gene that lacks one α ₂ gene, which is the detection result of α thalassemia 2 patients, and line 7 is the deletion of two α genes (α ₁ and α ₂ ) The amplified electrophoresis band of the DNA genome, which is the test result of α-thalassemia 1 patient, row 8 is the amplified electrophoresis band of the DNA genome missing three α genes, which is the test result of the HbH patient, row 8 9 is the amplified electrophoresis band of the DNA genome with deletion of all 4 α genes, which is the detection result of a patient with Bart's edema.

The sequence of the primers of the present invention is clear, and can be automatically synthesized by a DNA synthesizer. It is routine and easy to perform in this research field. The gene amplified by the primers is clear. It is equipped with the amplification reagent system, amplification program and electrophoresis detection of the present invention, so that the results can be displayed. Clearly.

The present invention can not only detect whether the _α1 gene and the _α2 gene are missing, but also compare the internal reference standard of normal people to accurately determine whether the detected human hemoglobin (globin) α gene deletion belongs to the _α1 gene and the _α2 gene. Genes are also homozygous or heterozygous for the deletion of the _α1 gene or the _α2 gene. Reliably perform typing detection for the deletion of human hemoglobin (globin) alpha gene, without false positive or false negative error detection.

The technology of the invention is equipped with a detection kit made by the technology, which is convenient to operate and has clear detection results, and is convenient and reliable for both scientific research and α gene deletion typing detection.

Fig. 1 is the electropherogram of the PCR typing detection of human hemoglobin (globin) α ₁ gene and α ₂ gene.

Fig. 2 is a PCR genotyping detection chart of a family with a progenitor of HbH disease.

Example: In a 50μl system, containing 10mM Tris-HCl (PH=8.5), 50mM KCl, 2mM MgCl ₂ , 10mN(NH ₄ ) ₂ SO ₄ , gelatin 200μg/ml, formamide 8%, dNTP each 200μM, 0.25μM αp ₁ , 0.125 μM αp ₂ , 0.125 μM αp ₁ , 0.125 μM βp ₁ , 0.125 μM βp ₂ , about 0.5 μg of human chromosomal DNA, and cover the liquid surface with 30 μl liquid wax. Mix the reaction system well, centrifuge at a high speed for 30 seconds, add 1U of DNA polymerase in a constant temperature water bath at 93°C for 1 minute, mix well, centrifuge, and then proceed in a constant temperature water bath at 93°C for 1 minute, 55°C for 1 minute, and 68°C for 2 minutes, in sequence After 35 cycles, the cycle was completed and then incubated at 72°C for 5 minutes, 20 μl of the reaction product was electrophoresed on a 1.5% agarose gel (containing 0.5 μg/ml EB), and the electrophoresis band was observed under ultraviolet light.

This subject is an example of PCR genotyping detection in a family with a progenitor of HbH disease. The results of the observed electrophoresis bands are shown in Figure 2. The brightness of the second electrophoresis band in line 6 (father of the pioneer) in the figure, that is, the 603bp band of the amplified _Hbα2 gene fragment of αp ₁ and αp ₂ , is only half of the brightness of the third β band, so it can be concluded that The father is heterozygous for the deletion of the _α2 gene, and the father suffers from α2 syndrome. Line 7 is the test result of the mother of the pioneer. It can be seen that the brightness of the first and second electrophoretic bands are both less than half of the brightness of the third β band. It can be concluded that the mother is missing two α, α ₁ and α ₂ Gene (α ₁ and α ₂ are heterozygous), thus suffering from α to 1 syndrome. The electrophoretic bands of the pioneer himself are shown in row 8. There is no α ₂ (603bp) electrophoretic band, and the brightness of the α ₁ (278bp) electrophoretic band is only half of the brightness of the β (400bp) electrophoretic band. It can be concluded that it is missing Three α genes, suffering from HbH disease.

Claims (1)

1. one kind with archaeal dna polymerase chain reaction technology, to human hemoglobin alpha ₁Gene and α ₂Genetically deficient is carried out the gene branch

The Genetic Detection method that type detects is characterized in that:

(1) Oligonucleolide primers of gene amplification and order thereof are:

αp ₁：5’-CGG?CTC?TGC?CCA?GGT?TAA?GG-3’

αp ₂：5’-CCT?TGG?TCT?GAG?ACA?GGT?AAA?CA-3’

αp ₃：5’-AGT?GGG?GCC?GAG?GGC?CCA?G-3’

βp ₁：5’-AAG?GAG?ACC?AAT?AGA?AAC?TGG?GC

βp ₂：5’-TCT?CCC?CTT?CCT?ATG?ACA?TGA?AC；

(2) primer α p ₁With α p ₃Amplification Hb α ₁Gene fragment 278bp, α p ₁With α p ₂Amplification Hb α ₂Gene fragment

603bp, β p ₁With β p ₂Amplification Hb beta gene fragment 400bp;

(3) the gene amplification reagent system is to contain in 30-100 μ l reaction system:

Tris-HCl：10-50mmol/L?pH=8.1-8.5；KCl：50mmol/L；MgCl ₂：0.5-3.0mmol/L；

(NH ₄) ₂SO ₄: 10-25mmol/L; Gelatin: 200 μ g/ml; Methane amide: 4-8%; DNTPs:200-300

Mmol/L; Hot resistant DNA polymerase: 1-1.5U;

αp ₁：0.25-0.50μmol/L；αp ₂：0.125-0.25μmol/L；αp ₃：0.125-0.25μmol/L；

βp ₁：0.125-0.25μmol/L；βp ₂：0.125-0.25μmol/L；

(4) program of amplified reaction is:

1. 90-93 ℃ sex change 3-10 minute, enzyme-added beat even, centrifugal;

2. press 90-93 ℃ 0.5-1 minute, 50-60 ℃ 0.5-1 minute, 68-72 ℃ 1-2 minute, be 30-35 and follow

Ring:

3. 68-72 ℃ of insulation is after 3-7 minute, and negate answers product containing electrophoresis on the sepharose of EB, ultraviolet

Lamp is observed electrophoresis band down:

(5) detected characteristics is:

1. if inspected human hemoglobin alpha gene is α ₁Gene or α ₂The heterozygote of genetically deficient, then its α ₁Base

Cause or α ₂The brightness of the amplified production electrophoresis band of gene fragment only is the bright of oxyphorase β gene electrophoresis band

Spend below half:

2. if inspected human hemoglobin alpha gene is α ₁Gene or α ₂The homozygote of genetically deficient, then amplified production

During electrophoresis, the β genonema is arranged, do not have α ₁Genonema or α ₂Genonema;

3. if inspected human hemoglobin alpha gene has lacked 3 α genes, then α ₁Gene or α ₂Gene fragment expands

Volume increase thing electrophoresis band has only one, and its brightness only is that the brightness of β genonema is below half;

4. if lacked 4 α genes in the inspected human hemoglobin alpha gene, the β genonema is only arranged then, do not have α ₁

Genonema and α ₂Genonema.

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