RU2484445C2 - Method for sampling of bone material for paleogenetic, biochemical and radiocarbon survey - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method for sampling of bone material for paleogenetic, biochemical and radiocarbon survey includes drying of bone material and making bone specimens. Specimens are produced by sawing of bone material into plates with subsequent polishing and mechanical extraction of non-fossilised bone substance. Extraction of non-fossilised bone substance is carried out in a dark room, being illuminated with a long-wave ultraviolet luminescent lamp with wave length of 340-380 nm.

EFFECT: improved quality of specimen preparation, making it possible to extract non-fossilised bone sections from bone samples, increased validity of survey.

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Description

The invention relates to biology, namely to paleogenetics. Promising is its use in forensic practice in the identification of bone remains and in archeology in radiocarbon dating of bone material.

Bone remains can persist in the soil for a long time. They are widely used in biochemical and genetic research, in forensic practice in the genetic identification of bone remains, as well as in archeology in determining the absolute age of archaeological materials by radiocarbon analysis. It is known that fossilization (natural mineralization) significantly reduces the accuracy of such studies.

To reduce the negative consequences of fossilization, the study uses long tubular bones, the least susceptible to fossilization, the bone material of which is subjected to special chemical treatment (Petrishchev V.N., Kutueva AB, Rychkov Yu.G. A simple and effective method for isolating DNA from fossils bones for subsequent amplification using polymerase chain reaction // Genetics. - 1993. - T.29. - No. 4. - S.690-191; DM Mognonov et al. Method for absolute dating of osteological material from archaeological sources. RF patent No. 2187096 about t 10.08.02; Mognonov D.M. et al. Method for dating osteological material of archaeological sources by pyrolytic gas chromatography. Patent of the Russian Federation No. 2194980 dated 12/20/02.) But existing methods of sample preparation do not completely exclude the inclusion of fossilized bone material into the studied samples , which significantly reduces the accuracy of subsequent studies.

The purpose of this invention is to improve the quality of sample preparation, allowing you to select from the bone samples not fossilized areas of bone suitable for further biochemical, genetic and radiocarbon studies.

Many proteins, including ossein, a bone substance, are capable of photoluminescence under the influence of long-wave ultraviolet radiation with a wavelength of 315-400 nm, which disappears when it is denatured. (Chernogryadskaya N.A. et al. Ultraviolet Fluorescence of a Cell. - L .: Nauka, 1978.) Traditionally, the Wood Lamp “black lamp” with a wavelength of 340-380 nm is used to study biological materials. A factor hindering photoluminescence is also the presence on the sample of a large amount of inorganic contaminants, including water or particles of abrasive material. The use of harder ultraviolet radiation during the preparation of biological objects can lead to protein degradation and is hindered by the need to use special protective equipment (GOST 4557-88 Sanitary norms of ultraviolet radiation in industrial premises). Ultraviolet radiation with a wavelength greater than 380 nm is not used, as it is perceived by the human eye as weak light and mask luminescence.

This goal is achieved by the fact that the preparation of bone samples is carried out under illumination with a long-wave ultraviolet fluorescent lamp with a wavelength of 315-400 nm.

At the first stage of sample preparation, bone samples are made, for which the bone material is pre-dried, then sawn into 5-7 mm thick plates. The thickness of the plates is determined by the convenience of further preparation: in the case of using thinner samples, the likelihood of their damage during grinding increases, the use of thicker samples creates inconvenience in the selection of the necessary samples. The resulting samples are ground on abrasive wheels of decreasing grit until scratches disappear visible to the naked eye. Further sample preparation is carried out in a darkened room when illuminated with an ultraviolet fluorescent lamp with a wavelength of 315-400 nm. Under such lighting, a non-fossilized bone acquires a bright bluish-white color, a fossilized bone - a dull grayish, brownish, sometimes almost black, particles of abrasive materials - a light purple. Particles of abrasive materials are removed from the surface with a natural bristle brush. Further selection of a non-fossilized bone fragment is carried out in any mechanically accessible way: with a milling cutter, a jigsaw, or with a scalpel.

We have studied long tubular bones from 37 burials of the 17th century on the territory of Smolensk. Figure 1, 2 presents a photograph of a section of the tibia of an adult. The thickness of the section is 7 mm. In Fig.3, 4 is a photograph of a thin section of a large femur of a child. The thickness of the section is 5 mm. Sections were obtained by sawing with a jigsaw and then polishing on abrasive wheels with decreasing grain size, then they were photographed under visible light and under illumination with a long-wave ultraviolet fluorescent lamp.

The proposed method for the isolation of non-fossilized bone leaves allows one to obtain the most preserved fragments of bone material for research, which sharply increases the accuracy of biochemical, paleogenetic, radiocarbon studies, and increases the reliability of identification of bone remains in forensic medicine.

The method is easy to use, based on the use of available equipment, does not require special training of personnel.

The invention is illustrated by figures, where Fig. 1 shows a photograph of a bone specimen of the tibia of an adult from a burial place of the 17th century under visible lighting, Fig. 2 shows a photograph of a bone specimen of a tibia of an adult from a burial of the 17th century under ultraviolet light of a long-wave ultraviolet luminescent a lamp with a wavelength of 315-400 nm, position 1 indicates non-fossilized bone substance; position 2 - fossilized bone substance; position 3 - the area slightly contaminated with abrasive material. Figure 3, 4 presents a photograph of a bone sample of the femur of a child under visible lighting and illumination with a long-wave ultraviolet fluorescent lamp with a wavelength of 315-400 nm.

Claims (1)

The method of sampling bone material for paleogenetic, biochemical and radiocarbon studies, including drying the bone material, manufacturing bone samples obtained by cutting bone material into plates, followed by grinding and mechanical isolation of unfossilized bone substance, characterized in that the selection of unfossilized bone substance is carried out in a darkened room when illuminated with a long-wave ultraviolet fluorescent lamp with a wavelength of 340-380 nm.

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