The way to determine the genetic stability of the subject to infection by the human immunodeficiency virus first type (hiv-1)

 

(57) Abstract:

The method is designed to determine the genetic stability of the subject to infection by the human immunodeficiency virus. The method involves isolation of the DNA of the subject, the amplification of the structural gene co-receptor CCR-5 and carrying out polymerase chain reaction. Next, perform the determination of the size of the resulting amplified fragments and detection of deletion mutations. Using the primers of the following structure:

5' GGGTGGCTGTGTTTGCG3'

5' ACCATGACAAGCGGCAGT3'.

The proposed method allows to speed up and simplify the process of determining the stability of the subject to infection. 2 C.p. f-crystals, 2 Il.

The invention relates to the field of biotechnology, genetic engineering and medical genetics and can find application in medicine in the diagnosis of hereditary genetic stability of the individual to infection by the human immunodeficiency virus type 1 (HIV-1) and the development of acquired immunodeficiency syndrome (AIDS).

It is known that HIV-1 infects CD4+ cells. In the mechanism of infection involved the used and when their deficiency virus HIV-1 is not able to penetrate into CD4+ cells and multiply in organisator SSC-5 (Science, 1996, V. 273, No. 5283, p. 1797) [1].

It is known that the structural gene for the CCR5 receptor is located on chromosome R. Described deletion mutation in this gene 32 base pairs (p. O. ), resulting in impaired development of the HIV-1 virus in CD4+ cells (Science, 1996, V. 273, No. 5283, p. 1856-1862) [2]. It is known that homozygosity for this deletion leads to a complete genetic resistance to HIV-1 virus, and heterozygosity for a deletion in the gene CRC5 receptor leads to slower progression of HIV-1 infection and easier clinical course of AIDS in persons infected with HIV-1 (Nature. 1996, V. 382. R. 722-725) [3].

There is a method of determining the genetic stability of the subject to infection by the virus HIV-1 by detection of deletion mutations in the structural gene for the CCR5 receptor, including DNA isolation of the subject, the amplification of fragments of the structural gene by polymerase chain reaction (PCR) using two primers complementary to the two sections of the structural gene, located by respectively 5' and 3' ends of detectable deletion mutations, restrictiona amplified DNA fragments by the enzyme Eco RI and determining the length of the obtained amplified fragments and detection of dealswith primers, which are complementary to such DNA regions of the gene CCR5, which are located at a considerable distance from the detected deletion mutations. The consequence of this is that in the PCR receive the amplified DNA fragments of considerable length (about 800 p. N.). As a result of this amplificatoare DNA fragments must be subjected before electrophoresis of restriction, which increases labour costs and the consumption of reagents. The result of the restriction is to obtain a sufficiently large size of the DNA fragments by size 371-403 p. N. Accuracy of detection of a short section of the deletion size 32 p. N. in such large fragments is low. In addition, in the analysis method prototype use isotopomer connection that leads to an increase in the duration of the analysis and increases the risk of personnel exposure and environmental contamination by radioactive isotopes.

The technical result obtained by implementing the present invention is the simplification and acceleration determination that the subject of homozygosity or heterozygosity for the deletion of 32 p. O. gene of CCR5 co-receptor, increasing its reliability, opt isotopomer connections.

Achieve-1 through the identification of deletion mutations in the structural gene for the CCR5 coreceptor, including DNA isolation of the subject, the amplification of the fragment of the structural gene by PCR using two primers complementary to the two sections of the structural gene, placed by 5' and 3' ends of detectable deletion mutations, determining the size of the resulting amplified fragments and detection of deletion mutations on the results of determination.

A distinctive feature is that using the primers:

5' GGGTGGCTGTGTTTGCG 3' (PR1)

5' ACCATGACAAGCGGCAGT 3' (NP2)

In a specific implementation, conduct preliminary processing of DNA examined by heating for 4.5-5.5 minutes at 94-96oC, and then spend 25-35 cycles of PCR. Each cycle consists of a serial thermal processing for 45-70 seconds at a temperature 93-96oC, 63-66oC and 70-73oC, after which the cycle repeats. The length of the amplified fragments is recommended to determine electrophoresis in 4-6% polyacrylamide gel followed by staining zones localization of amplified DNA fragments bromide by ethidium or silver nitrate.

Unlike prototype [3] in the new method first used the new primers, complementary to those areas I mutations. This allows to obtain PCR short amplificatoare fragments of a structural gene that does not require their restriction in the future - and therefore no need to use expensive restrictase and the need for labor costs for conducting this phase of the determination. The small size of the amplified fragments of the structural gene allows to speed up and simplify the process of determining their size and to refuse the use of isotopomer connections.

In Fig. 1 presents the structure of the CCR5 gene in the region of deletion mutations with relative localization of the site of deletion of the newly proposed in this primer.

In Fig. 2 shows the detection results of the examined individuals of deletion mutations in the structural gene CCR5.

Below are examples of the implementation of the new method.

Example 1.

For genotyping by deletions in the structural gene for the CCR5 receptor using DNA isolated from peripheral blood or from cells of the epithelium of the mucous membrane of the oral cavity of the subject.

For DNA extraction from blood 20 ml of blood obtained from a finger, literally in 500 ál of bidistilled water and the nucleus of lymphocytes was resuspendable in 100 μl of a 5% solution of the resin Chelex-100 (Bio-Rad), warmed up for 30 minutes at 56oC and 8 minutes at 95oC. After centrifugation was performed for 5 minutes at 12000 g and 5 μl of the supernatant (selected DNA) were introduced into a test tube with 15 μl of buffer for polymerase chain reaction for thermal denaturation of DNA and polymerase chain reaction.

For DNA extraction from cells of the epithelium of the oral cavity the mouth was rinsed with 20 ml of distilled water and 1.5 ml of the obtained flushing centrifuged for 10 minutes at 12000 g. The supernatant was collected, the precipitated cells were washed twice in 1 ml of buffer TE (pH 8.0) and then added to the precipitate 100 μl of a 5% solution of Chelex-100. Next, DNA was isolated as well as from peripheral blood cells.

Example 2.

After the initial thermal denaturation selected DNA for 5 minutes at 94oC was added 1 unit of Taq DNA polymerase (manufactured by the Institute of molecular genetics of RAS, Russia) and carried out 30 cycles of polymerase chain reaction in the following mode: 1 minute at 95oC, 1 minute at 64oC and 1 minute at 72oC.

For amplification of carrying a deletion of 32 p. O. plot structural gene CCR5 receptor were used new primers GGGTGGCTGTGTTTGCG (PR1) and ACCATGACAAGCGGCAGT (NP2), which complementary. and 27 p. O.

After 30 cycles of polymerase chain reaction, the reaction mixture was incubated 10 minutes at 72oC and cooled to 4oC.

Example 3.

Obtained in the process of polymerase chain reaction amplificatoare fragments of the structural gene CCR5 person analyzed without any additional processing by electrophoresis in 5% polyacrylamide sedentarism gel size 200 mm x 160 mm x 0.5 mm After conducting electophoresis gel was stained with a solution of ethidium bromide in distilled water with a concentration of 10 µg/ml was Found that in the case of the gene CCR5 receptor without deletions amplificatory fragment has a size of 175 p. O., in the case of the gene CCR5 receptor with a deletion of amplificatory fragment has a size of 143 p. O.

Example 4.

According to the method of examples 1-3 analysis of peripheral blood samples from a group of the subject of 9 people resistant to HIV-1 infection. The results are presented in Fig. 2. Found 2 persons with partial resistance to HIV-1 infection, 7 susceptible to HIV infection people. We are sensitive to HIV-1 infection is detected only fragment size 175 p. O. In persons with partial sites for Dr HIV-1 infection WIEGO time while the definition by a known method requires two working days.

1. The way to determine the genetic stability of the subject to infection by the human immunodeficiency virus first type (HIV-1) through the identification of deletion mutations in the structural gene for the CCR5 coreceptor, including DNA isolation of the subject, the amplification of the structural gene CCR5 coreceptor by carrying out thermal denaturation of DNA and subsequent polymerase chain reaction using two primers complementary to the two sections of the structural gene is located from the side, respectively, with 5' and 3' sides of the detected deletion mutations, determining the size of the resulting amplified fragments and detection of deletion mutations on the results of the determination, wherein using the primers of the following structure:

5'GGGTGGTGTGTTTGG3'

ACCATGACAAGCGGCAGT3'.

2. The method according to p. 1, wherein the preliminary heat treatment of the DNA of the subject is carried out by heating for 4.5 to 5.5 min at 94 - 96oC, and then spend 25 to 35 cycles of PCR, each cycle consists of a serial thermal processing within 45 70 at a temperature of 93 - 96oC, 63 - 66oC and is the amplified fragments detected by electrophoresis in 4 - 6% polyacrylamide gel followed by staining zones localization of amplified DNA fragments bromide by ethidium or silver nitrate.

 

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