Use, method and kit of polymorphism locus gene type predict agiotensin converter enzyme inhibitor

Abstract

Use of polymorphism site gene to predict ACEI medicine, its method and reagent kit are disclosed. It can be used to determine critical enzyme gene polymorphism site gene typing oligonucleotide and critical enzyme gene polymorphism site gene in cysteine metabolic pathway.

Description

technical field

[0001] The invention relates to the use of polymorphic site genotypes of key enzyme genes on the homocysteine ​​metabolic pathway for predicting the effect of angiotensin-converting enzyme inhibitors (ACEI) drugs. The present invention also relates to a polymorphism typing oligonucleotide for determining the polymorphic site genotype of key enzyme genes on the homocysteine ​​metabolism pathway, a polymorphism typing oligonucleotide for determining homocysteine ​​metabolism The polymorphic site genotype of the key enzyme gene on the pathway, the method and kit for predicting the action effect of drugs containing angiotensin converting enzyme inhibitors. The invention belongs to the field of medicine. Background technique

[0002] Hypertension is one of the most common chronic diseases in my country and the world. The global incidence of hypertension is as high as 31.3%, about 690 million people. Hypertension is the main cause of life-threatening heart and c...

Examples

Embodiment 1

[0094] Example 1: Determining the C677T (Ala222Val, dsSNP ID: rs1801133) polymorphism site of the MTHFR gene and predicting the effect of drugs containing ACEI

[0095] (1) Determination of the C677T polymorphism site genotype of the MTHFR gene

[0096] (1) Extract the genomic DNA of the host cell

[0097] (a) Add 30ml of erythrocyte lysate to the whole blood, shake slowly, and let stand at room temperature for 10 minutes. During this period, shake several times to completely lyse the erythrocytes;

[0098] (b) Centrifuge at 4°C, 2000 rpm for 10 minutes, remove the supernatant, break up the precipitated leukocytes on a rotary shaker, add 40ul of protease and 50ul of RNase, shake well, add 15ml of leukocyte lysate, mix Take it out after evenly bathing in 37°C water bath for 20 minutes, and put it in cold water;

[0099] (c) Add 4ml of cold protein precipitation solution, mix well and place in -20°C refrigerator for 5 minutes, take it out and centrifuge at 3000 rpm for 10 minu...

Embodiment 2

[0184] Example 2: Determining the A1298C polymorphism site (Glu 429Ala, dbSNP ID: rs1801131) of the MTHFR gene and predicting the effect of drugs containing ACEI

[0185] (1) Determination of the genotype of the 1298AC polymorphism site of the MTHFR gene

[0186] (1) According to the conventional operation process, adopt the method similar to Example 1 to extract the genomic DNA of the host cell

[0187] (2) Use the Taqman method to detect the genotype of the 1298AC polymorphism site of the MTHFR gene

[0188] (a) Amplify the MTHFR functional gene polymorphic site and its flanking sequences with a PCR instrument, containing 10ng of genomic DNA in a 5ul PCR reaction system, and 2.5ul of Taq man 2XUniversal PCR Master Mix No AmpErase UNG (composition includes: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, PassiveReference, optimized buffer), and 0.72uM forward primer, 0.72uM reverse primer and two allele-specific probes with fluorescent reporter groups each 0.16uM.

[0189] T...

Embodiment 3

[0225] Example 3: Determination of MTRR His595Tyr (C1783T, dbSNP ID: rs10380) polymorphism site and prediction of the effect of drugs containing ACEI

[0226] (1) Determination of the genotype of the His595Tyr (CT) polymorphic site of the MTRR gene

[0227] (1) Extract the genomic DNA of the host cell:

[0228] On the basis of standard operating procedures, the genomic DNA in salivary cells was extracted by salt precipitation. DNA was stored at -20°C.

[0229] (2) The genotype of MTRR His595Tyr (C1783T) polymorphism was detected by TaqMan method.

[0230] (a) Amplify the polymorphic site of the MTRR His595Tyr gene and its flanking sequences with a PCR instrument, and contain 10ng of genomic DNA in a 5ul PCR reaction system, and PCR Master Mix No AmpErase UNG of 2.5ul TaqMan2X Universal (the composition includes: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, PassiveReference, optimized buffer), and 0.72uM forward primer, 0.72uM reverse primer and two allele-specific probes ...

technical field

[0001] The invention relates to the use of polymorphic site genotypes of key enzyme genes on the homocysteine ​​metabolic pathway for predicting the effect of angiotensin-converting enzyme inhibitors (ACEI) drugs. The present invention also relates to a polymorphism typing oligonucleotide for determining the polymorphic site genotype of key enzyme genes on the homocysteine ​​metabolism pathway, a polymorphism typing oligonucleotide for determining homocysteine ​​metabolism The polymorphic site genotype of the key enzyme gene on the pathway, the method and kit for predicting the action effect of drugs containing angiotensin converting enzyme inhibitors. The invention belongs to the field of medicine. Background technique

[0002] Hypertension is one of the most common chronic diseases in my country and the world. The global incidence of hypertension is as high as 31.3%, about 690 million people. Hypertension is the main cause of life-threatening heart and c...

Examples

Embodiment 1

[0094] Example 1: Determining the C677T (Ala222Val, dsSNP ID: rs1801133) polymorphism site of the MTHFR gene and predicting the effect of drugs containing ACEI

[0095] (1) Determination of the C677T polymorphism site genotype of the MTHFR gene

[0096] (1) Extract the genomic DNA of the host cell

[0097] (a) Add 30ml of erythrocyte lysate to the whole blood, shake slowly, and let stand at room temperature for 10 minutes. During this period, shake several times to completely lyse the erythrocytes;

[0098] (b) Centrifuge at 4°C, 2000 rpm for 10 minutes, remove the supernatant, break up the precipitated leukocytes on a rotary shaker, add 40ul of protease and 50ul of RNase, shake well, add 15ml of leukocyte lysate, mix Take it out after evenly bathing in 37°C water bath for 20 minutes, and put it in cold water;

[0099] (c) Add 4ml of cold protein precipitation solution, mix well and place in -20°C refrigerator for 5 minutes, take it out and centrifuge at 3000 rpm for 10 minu...

Embodiment 2

[0184] Example 2: Determining the A1298C polymorphism site (Glu 429Ala, dbSNP ID: rs1801131) of the MTHFR gene and predicting the effect of drugs containing ACEI

[0185] (1) Determination of the genotype of the 1298AC polymorphism site of the MTHFR gene

[0186] (1) According to the conventional operation process, adopt the method similar to Example 1 to extract the genomic DNA of the host cell

[0187] (2) Use the Taqman method to detect the genotype of the 1298AC polymorphism site of the MTHFR gene

[0188] (a) Amplify the MTHFR functional gene polymorphic site and its flanking sequences with a PCR instrument, containing 10ng of genomic DNA in a 5ul PCR reaction system, and 2.5ul of Taq man 2XUniversal PCR Master Mix No AmpErase UNG (composition includes: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, PassiveReference, optimized buffer), and 0.72uM forward primer, 0.72uM reverse primer and two allele-specific probes with fluorescent reporter groups each 0.16uM.

[0189] T...

Embodiment 3

[0225] Example 3: Determination of MTRR His595Tyr (C1783T, dbSNP ID: rs10380) polymorphism site and prediction of the effect of drugs containing ACEI

[0226] (1) Determination of the genotype of the His595Tyr (CT) polymorphic site of the MTRR gene

[0227] (1) Extract the genomic DNA of the host cell:

[0228] On the basis of standard operating procedures, the genomic DNA in salivary cells was extracted by salt precipitation. DNA was stored at -20°C.

[0229] (2) The genotype of MTRR His595Tyr (C1783T) polymorphism was detected by TaqMan method.

[0230] (a) Amplify the polymorphic site of the MTRR His595Tyr gene and its flanking sequences with a PCR instrument, and contain 10ng of genomic DNA in a 5ul PCR reaction system, and PCR Master Mix No AmpErase UNG of 2.5ul TaqMan2X Universal (the composition includes: AmpliTaq Gold DNA Polymerase, dNTPs with Dutp, PassiveReference, optimized buffer), and 0.72uM forward primer, 0.72uM reverse primer and two allele-specific probes ...