UA48849A - Method for isolation of dna/rna from biologic materials - Google Patents

Abstract

The method for isolation of DNA/RNA from biologic materials provides for employing NaX as the sorbent for nucleic acids.

Description

Description of the invention

The invention belongs to medicine, and more specifically to molecular biology and can be used for 2 isolation of DNA/RNA from biological and clinical material.

It is known that modern molecular biological research is inextricably linked with methods based on the amplification of nucleic acids.

Simple methods of extracting DNA from samples are necessary for a high-quality process of nucleic acid amplification. The main criteria when choosing a method are simplicity and speed, in combination with obtaining a sufficiently high amount of DNA, at the same time as successfully eliminating the possible presence of amplification inhibitors.

There are a number of methods for DNA/RNA isolation. These include methods of phenol-chloroform deproteinization, digestion with proteinase K, boiling of samples, detergent treatment, a method using guanidine isothiocyanate together with silica gel (Maniatis T., Frich E.E., Szozmbruk J. / Methods of genetic engineering. Molecular cloning / / Edited by A.A. Baeva - M.: "Mir", 1984. - 480 p.). 12 Among the disadvantages of the detergent method and boiling is the probability of a high percentage of inhibitors in preparations, the proteinase K digestion method is quite expensive, when using phenol-chloroform extraction there is a high probability of nucleic acid loss during the washing steps (OSgeepPeyj 1... MUpNe T.0. / Zatrie rgeragayop teiodv. 1993. - R. 122 - 137. Ip O.M. Regvipd ei ai.

OiIadpovis toiesciag ptisgorioiodu: rgupsirie apa arriisaiopv. Ategisap bosieiu oi Misgobioiodu. 1997. - Z5. 20. Mo, 10, - R. 2628 - 2633).

Compared to the described methods, the method using guanidine isothiocyanate and silica gel makes it possible to obtain sufficiently high-quality preparations with a relatively small risk of DNA loss during the washing steps.

This method is recommended for the isolation of DNA/RNA for the further use of drugs in diagnostic and research activities (Votap .., Saudov S., Otsipp o T.S. / Moiiiesciag aiadpoviz oi Spiatuaia rpeshtopiae IpGesiop // .). Sp. Misgobio!. - 1999. - 68. - R. 3791 - 3799). "

The method of DNA/RNA extraction using guanidine isothiocyanate and silica gel is the closest to the claimed method, so it was chosen as a prototype. When using the prototype, silica gel is used as a nucleic acid sorbent. However, in some cases, when the amount of material is small, there is a partial loss of DNA, which can later, for example, when using the drug in the polymerase chain reaction (PCR), lead to a false-negative result (UMiivop R.A., U. RiIrre, O. Zatiei, Zatspdeg "zh

MA. / ЮОемеиоретепи ои зутрийНей роПтегазе сайп геасПоп-еплуте Ітипеазау иог (Ше дейесцопо ой

Spiatua|a rpeshtopiae IpGesi(op // -). Arri. Wasiegio!. - 1996. - 80: 431 - 438). co

The invention is based on the task of improving the quality of isolated DNA and preventing its loss. with

The problem, which is the basis of the invention, is solved by the fact that in the known method of DNA/RNA isolation, which includes the use of a sorbent, according to the invention, Mach zeolite is used as a sorbent. M

The features of the invention that distinguish it from the prototype - the use of silica gel - are the use of zeolite Mach as a nucleic acid sorbent.

Isolation of DNA by the guanidiniisothiocyanate zeolite sorption method was carried out according to the protocol. "

300 ml of 6M C 50 guanidine isothiocyanate and 1 ml of aqueous zeolite suspension were added to 100 ml of physiological solution containing the material under study. The samples were incubated for 10-15 minutes at room temperature, periodically shaking on a vortex. ZOS was centrifuged at 11,000 rpm. The supernatant was discarded.

15O0μl of 4M guanidine isothiocyanate was added to the precipitate, vortexed, and centrifuged at 11,000 rpm. The supernatant was discarded. 700 μl of washing solution (50905 ethyl alcohol, 0.05 M Mass and 0.01 TgizNHCI) was added to the precipitate. Vortex and centrifuge ZOs at 11,000 rpm. The supernatant was discarded.

The washing procedure was repeated twice. The samples were dried in a thermostat at 45 - 5573 for 5 minutes. 50 ml of deionized water was added to the precipitate and vortexed. Incubated for 5 minutes at a temperature of 45 - 557C,

Ge | centrifuged 15 - ZOs at 11,000 rpm. The supernatant containing DNA was transferred to a separate tube and used in PCR. for Preparation of an aqueous suspension of zeolite Mach. "iz" 20 god of zeolite Mach was ground in 500 ml of bidistilled water, allowed to stand for 10 minutes, 400 ml of supernatant was taken, bObOmcl of concentrated NOSE was added to it, stood for 24 hours, the supernatant was drained. The precipitate was weighed in 100 ml of H2oO and autoclaved.

The presence of a cause-and-effect relationship between essential features and achieved technical results is confirmed by the data of an experimental study, which showed that the introduction of a new sorbent 29 improves the quality of DNA preparations and prevents its loss during sample processing. in. To determine the ability of MaX zeolite to influence the quality of DNA isolated from clinical samples, nucleic acid preparations obtained using the prototype, as well as the new sorbent, were analyzed by PCR.

DNA was isolated in two ways from urogenital scrapings, the obtained preparations were subsequently used for PCR. PCR was performed using three pairs of primers selected for amplification of DNA fragments of SpPpiatua|a igaspotaiiv, Negrez Zitrieh Migiz type I, Suiotedaiomigiv. Amplification and registration of results was carried out in accordance with the recommended conditions (Unification of laboratory research methods in the diagnosis of STDs, Ministry of Health of Ukraine, 2000).

Table 1 shows the PCR results obtained on clinical samples from which DNA was isolated by the prototype method and the new method.

Table 1

Results of PCR on DNA preparations obtained using different sorbents

Negreg vitrivkh motif 1 type suotevayutte| 77771112 61611 и и и и и ше и

Note: the number of " corresponds to the number of inhibited samples.

The obtained results indicate that the introduction of Mach zeolite into the composition of reagents for DNA/RNA isolation has a positive effect on the quality of the obtained preparations. The number of positive PCR results when using the new isolation method increases, which can be explained by the better sorbing properties of Mach zeolite.

The structure of zeolite MaX promotes more reliable binding of DNA molecules on its surface, so that the risk of DNA loss is reduced during the washing steps. The number of inhibited samples when using zeolite is smaller than when using silica gel. It can be assumed that low molecular weight substances, among which PCR inhibitors may be present, are sorbed on the porous structure of zeolite Mach and, therefore, their concentration in the preparations of the selected

DNA decreases.

Thus, the proposed method allows obtaining higher quality DNA preparations, preventing its loss.

Claims (1)

The formula of the invention is the method of extracting DNA/RNA from biological material, which includes the use of a sorbent of nucleic acids, which differs in that zeolite Mach is used as a sorbent. "Official Bulletin "Industrial Property". Book 1 "Inventions, useful models, topographies of integrated microcircuits", 2002, M 8, 15.08.2002. State Department of Intellectual Property of the Ministry of Education and Science of Ukraine. " (ee) (ee) " - and? Chek" (ee) (ee) Chek" 3e) 60 b5