Isotopic tracer method (isotopic tracer method) is a microanalytical method that utilizes radionuclides as tracers for labeling of research objects, and the creator of the tracer experiment was Hevesy.Hevesy was the first to use naturally occurring radioactivity 212Pb to study the distribution and translocation of lead salts in the distribution and translocation of lead salts in legumes. The discovery of artificial radioactivity by Jolit and Curie in 1934 and the subsequent establishment of production methods (gas pedals, reactors, etc.) provided the basic conditions and strong guarantee for the faster development and wide application of radioisotope tracing.
I. Basic principles and characteristics of isotope tracing method
The radionuclides (or stable nuclides) and their compounds utilized in isotope tracing have the same chemical and biological properties as those of the corresponding common elements and their compounds existing in the natural world, and they only have different nuclear physical properties. Therefore, isotopes can be used as a kind of marker to make isotope-containing labeled compounds (e.g., labeled foods, drugs, and metabolic substances, etc.) instead of the corresponding non-labeled compounds. The use of radioactive isotopes constantly emit characteristic rays of nuclear physical properties, you can use a nuclear detector at any time to track its location in the body or outside the body, the number and its transformation, stable isotopes do not emit rays, but it can be used with the corresponding isotopes and the mass of the ordinary difference between the quality of the instrument through mass spectrometry, gas chromatography, NMR *** vibration, and other quality analysis instruments to determine. Radioactive isotopes and stable isotopes can be used as tracers (tracer), however, stable isotopes as tracers its sensitivity is low, can be obtained in fewer types, the price is more expensive, the scope of its application has been limited; and the use of radioactive isotopes as a tracer is not only the sensitivity, the measurement method is simple and easy to carry out, can be accurately quantified, accurate localization and in line with the physiological conditions of the subject of the study and other features
1. High sensitivity
Radioactive tracer method can be measured to the 10-14-10-18 gram level, that is, from 1015 non-radioactive atoms can be detected in a radioactive atom. It is 108-107 times more sensitive than the more sensitive weight-analysis balances currently available, and the most accurate chemical analysis method to date is difficult to measure to the 10-12 gram level.
2. Simple method
Radioactivity determination is not subject to the interference of other non-radioactive substances, you can omit many complex material separation steps, in vivo tracing, you can use certain radioisotopes to release penetrating r-rays, and in vitro measurements to obtain the results, which greatly simplifies the experimental process to achieve a non-destructive analysis, with the development of liquid scintillation counting. With the development of liquid scintillation counting, radioisotopes that emit soft beta rays such as 14C and 3H are more and more widely used in medical and biological experiments.
3. Accurate positioning and quantification
Radioisotope tracer method can accurately and quantitatively determine the metabolic transfer and transformation of substances, and certain morphological techniques (such as pathology, tissue sectioning technology, electron microscopy technology, etc.), you can determine the quantitative distribution of radiotracer in the tissues and organs, and the positioning of the tissues and organs can be up to the level of the cellular level, the subcellular level, and even the molecular level. and even at the molecular level.
4. In line with physiological conditions
In the radioisotope experiments, the chemical amount of the quoted radioactively labeled compounds is extremely small, it is insignificant to the body of the original weight of the corresponding material changes, the body physiological processes still maintain a normal state of equilibrium, to obtain the results of the analysis of the physiological conditions, and more reflective of the objective existence of the essence of things. The advantages of radioisotope tracer method as mentioned above, but there are some defects, such as the personnel engaged in radioisotope work to be subjected to certain specialized training, to have the appropriate safety measures and conditions, at present, individual elements (such as oxygen, nitrogen, etc.) there is no suitable radioisotopes and so on. When making tracer experiments, must also pay attention to the tracer isotope effects and radiation effects. The so-called isotope effect refers to radioactive isotopes (or stable isotopes) and the corresponding common elements between the existence of small differences in chemical properties caused by the individual properties of the obvious difference, for light elements, isotope effect is more serious. Because the mass discrimination between isotopes is multiplicative, such as 3H mass is three times that of 1H, and 2H is twice that of 1H, when using tritium water (3H2O) as a tracer, its content in ordinary H2O cannot be too large, or it will make the physical constants of the water, the permeability of the cell membrane and the viscosity of the cytoplasm, etc., will be changed. However, in general tracer experiments, the error caused by the isotope effect is often within the experimental error and is negligible. Radioactive isotopes release rays for tracing measurements, but the role of rays on the organism reaches a certain dose, will change the physiological state of the organism, which is the radiation effect of radioisotopes, so the amount of radioisotopes should be less than the safe dose, strictly controlled in the biological organisms can allow the paradigm
Within the limits, so as to avoid the experimental object of radiation damage, and get the wrong results.
The design principle of tracer experiment
The design of a radioisotope tracer experiment should be considered from the purpose of the experiment, the conditions of the experiment and the level of protection against radioactivity. In principle, two main aspects must be designed for radioactive tracer experiments: one must seek effective and repeatable conditions for the determination of the intensity of radioactivity, and the second is to choose a suitable specific activity λqδ (units are atoms / time / molecules, dpm / mol or ci / mol). where λ = -dN'dt/N' is the decay constant of the radioactive nucleus at the site. q = N '/n', indicating that n' molecules of this chemical form are labeled by N' radioactive atoms. δ = n', indicating that n' molecules of this chemical form are labeled by N' radioactive atoms. δ = n'/n denotes the ratio of the number of radiolabeled molecules n' to the total number of molecules (labeled plus unlabeled) n. The use of radioisotope tracer technology to achieve all or part of the intended purpose of the research subject, generally through the experimental preparation stage, experimental stage and radioactive waste disposal of three steps.
(I) experimental preparation
1. Selection of tracer
The specific activity λqδ of the selected radiotracer must be large enough to ensure the sensitivity required for the experiment, but as small as possible, so that the radiation decomposition can be ignored under the experimental conditions. The general situation is to select radioisotopes with appropriate decay mode, radiation type and half-life, and low radiotoxicity, according to the purpose of the experiment and the length of the experimental period. The radionuclides identified so far include 58 naturally occurring and about 1,300 artificially produced ones, most of which are not often used as radiotracers. The main reasons for this are difficulties in preparation, unsuitable half-lives and insufficient radioactivity for quantification. In any production method, the production step is likely to involve more or less chemical treatment, and thus the tracer experimenter needs to know the chemistry of a nuclide and those elements surrounding it that may become impurities in the radioisotope.
Radioisotopes all decay (with or without an intermediate state) to a daughter nuclide in the ground state, and the decay is accompanied by various forms of energy radiation, such as alpha, beta-, beta+, gamma, and X-radiation. In the choice of tracer, tracer experimenters should carefully study the decay program map, according to the experimental conditions and counting conditions to decide that kind of radiation, in the decay program, on behalf of the nuclear energy level between the two horizontal lines and the distance between the energy difference, ↑ or ↓ indicates that the energy level with the same accompanied by the atomic number of increase or decrease in energy, ↓ indicates that from the excited state to the ground state of homogeneous and heterogeneous energy leaps. Generally, the most suitable half-life τ of the radioisotope should be selected, so that τ is long enough to make the decay correction meaningful or simply do not have to make the decay correction, and at the same time, it should be short enough to carry out tracer experiments in a safe manner, and make the radioactive waste easy to dispose of, in practice, the use of radioisotopes should be used with the half-life of the experiment needs to be continued for a period of time t to be compatible, such as for a certain experiment, t / τ = 0.04, should be selected for the half-life of the radioisotope. For example, for a certain experiment, t/τ=0.04, the decay of the selected radioisotope should be corrected to 3.5%; while t/τ=0.10, the decay of the selected radioisotope should be corrected to 6.6%. t/τ=0.15, it should be selected to be corrected to 10%.
In the in vitro tracer conditions, generally choose a longer half-life and moderate ray intensity, both for detection, but also easy to protect and preserve the radioactive tracer. In vivo tracer conditions, if the experimental cycle is short, should be selected with a short half-life, and can emit a certain intensity of r-ray material radioisotope, if the experimental cycle is long, such as the need to kill the animal live after the organization of organs were determined, should be selected with a longer half-life radioisotope. In addition, according to the purpose of the experiment to select the localization of the labeling tracer or not, for example, the study of amino acid decarboxylation reaction, 14C should be labeled in the carboxyl group, only this localization of the labeling of amino acids, in order to decarboxylation of the 14CO2 can be produced, while some experiments do not require a specific location of the labeling, but only uniform labeling can be.
The choice of radiotracer must also meet the requirements of high chemical purity and high radionuclide purity. In the tracer preparation period, storage period to use the test system in the use of solvents, chemical reagents, enzymes, etc. may produce chemical impurities, radiochemical impurities and radioactive impurities caused by self-decomposition of radiation, the presence of these impurities, so that tracer experiments in the use of tracers is not "pure", and more or less affect the results of the experiment, and even The presence of these impurities makes the tracer used in tracer experiments not "pure" and affects the results to a greater or lesser extent, or even leads to wrong conclusions. Tritium-labeled thymine nucleoside (3H-TdR) and uracil nucleoside (3H-UR) are two commonly used tracers, the former is effectively bound to the DNA, and the latter is doped into the RNA, and the rate of their radiolysis increases with the increase of the comparative radioactivity and the prolongation of the preservation time, and their stability is also different in different temperatures and different solutions. The stability of 3H-radiolysis increases with the increase of comparative radioactivity and the prolongation of preservation time. After eight years of storage about 35% of 3H-TdR is radiolyzed to 3H-thymine and results in the form of diols and hydrates, and in experiments this impurity is quickly incorporated into the cell and binds to macromolecules (most likely proteins) rather than to DNA and RNA, and these impurities are not removed by treatment of the cells with either DNAase or RNAase. 3H-TdR and 3H-TdR are not removed by treatment of the cells with DNAase or RNAase. -TdR and 3H-UR stored in a frozen solution at -20°C increase the rate of radiation separation by a factor of 3-4 over +2°C, but lower temperatures (-140°C) are also favorable for storage, and will be instructive in allowing for the tracer experimenter in choosing a radiotracer for preservation.
2. Choice of radioisotope measurement method
The choice of measurement method depends on the type of ray, for α-rays can usually be used for zinc sulfide crystals, ionization chambers, nuclear latex and other methods of detection; high energy β-rays can be used for mica window counting tubes, plastic scintillation crystals and nuclear latex determination, for the low-energy β-rays can be used to measure liquid scintillation counters: for γ-rays, they use G-M counting tube, sodium iodide (thallium) scintillation crystal detection. At present, most laboratories mainly use crystal scintillation counting and liquid scintillation counting method of measurement.
The same detection instrument for different amounts of tracer has different optimal working conditions, in the preparation stage of the experiment to check whether the detector has been adjusted to use the tracer isotope working conditions, otherwise you need to use a certain amount of tracer as a radioactive source (or the choice of the isotope of the standard source), the detector's optimal working conditions adjusted and to ensure that the detector's performance is in a stable and reliable state.
Detection
The best working conditions for the detection of the selection method: one is to measure the "ping curve", the other is to find the best quality factors. For photomultiplier tube, in theory there is no "ping" (plateau). However, with the increase of high voltage, within a certain range, the number of pulses change less, forming a small slope of the voltage pulse curve, usually also known as ping. The method of measuring the plateau curve: fix the radioactive source, according to the size of its ray energy, the initial selection of a wide range of instrument gain (amplification) and the threshold of the screener. Continuously change the high pressure (from low to high, uniformly increase the volts), every change in high pressure, are measured once the background and the count rate of the radioactive source, and finally make a high-pressure background count rate and high-pressure radioactive source count curve. With the same method, make another screening threshold (magnification unchanged) under the high pressure count rate curve, so that more than a few curves repeatedly. If necessary, the screening threshold can also be fixed, change the magnification, high pressure count rate curve. Should choose "ping" relatively flat curve working conditions: screening threshold and amplification gain, as a formal determination of the time of the instrument's working conditions, high pressure value should be selected in the "ping" mid-point of the corresponding high-pressure value of the side of the starting section. Quality factor, also known as the superior value, refers to a certain condition, to achieve the appropriate number of statistics required by the time is a function of the instrument's counting efficiency E and background count Nb: quality factor F = E2 / Nb it is a measure of the performance of a counter indicators, the instrument's quality factor F should be the larger the better, the larger the quality factor F, said that the measurement of the efficiency of E is the higher the background of the Nb is smaller. If the standard source of a particular radiotracer is difficult to source, etc., the relative quality factor f can be used instead. The relative quality factor f = ns/nb where ns is the count rate of a radioactive sample. The method of finding the best quality factor is the same as for ping curves, where several high-pressure-F (or f) curves are made, and the curve with the highest peak is chosen among the several curves. The conditions corresponding to the peak of this curve: high pressure, screening threshold, magnification, etc., are the optimal operating conditions of the instrument for the isotope being measured. The optimal quality factor does not necessarily fall exactly on the "ping", some are near the "ping" and some are at the lower end of the "ping". Tracer experimenters who focus on counting the entire energy spectrum of the isotope peaks advocate taking the working conditions corresponding to the "ping", while those who focus on the best value advocate taking the working conditions corresponding to the best quality factor, and there are also some people who make compromises. If the background of an instrument is very low, the noise of the photomultiplier is very low and the spectral resolution is high, there should not be much difference between the two. The optimal operating conditions for the same instrument change as the instrument's service life increases, and for different radioisotopes, the optimal operating conditions are different. Therefore, the best working conditions of nuclear detection instruments have exclusivity, and should often be selected through its different periods of the best working conditions. What's more, we can't use the same working conditions without asking the type of isotope to be measured.
In order to achieve accurate counting, can be a long time once counting, or a short period of time for many measurements, both of them to achieve the standard error is basically the same, in order to avoid the influence of external factors, in practice, take a short period of time for many measurements is more reasonable and applicable. When measuring the radioactivity of the sample, the background is an important influence factor. The background is high, the standard error and standard error are increased, especially in the sample count is low, the background of the standard error and standard error of the impact of the greater, thus affecting the accuracy of the results of the experiment, and in order to achieve a certain level of accuracy, it is necessary to increase the measurement time of the sample. According to the statistical law of nuclear decay, in the experiment if the number of samples is small, choose tN = 1.4tb ratio (where tN for the sample radioactivity measurement time, tb for the background measurement time) is more reasonable; if the number of samples is a large batch of samples, it is to prolong the background measurement time tb, take the average value of the time of tb, while the tN can be relatively short, which saves time and is conducive to shorten the experimental cycle. For tracer experimental design, the intensity of the radioactivity contained in the sample is required to make its radioactivity count rate is greater than or equal to 10-20 times the background count.
3. Non-radioactive simulation experiment, the whole process of the experiment to preview once
Isotope tracer experiments require accurate, careful, a little negligence or ill-considered in a hurry to carry out the formal experiment, not only is likely to lead to the failure of the experiment, but also lead to a waste of tracers and other experimental supplies, but also increase the radioactive waste, increase the level of the background of the laboratory, so that the experimentalist to accept unnecessary radiation dose, so the simulation experiment is not only the radioactive intensity of the sample is greater than or equal to the background count of 10-20 times. Radiation dose, so the simulation experiment can not only check the formal experiments in the equipment used, whether the drugs are qualified, but also operators can be trained to ensure that the formal experiments can be carried out smoothly.
(2) Formal experimental stage
1. Selection of radioisotope dose
The isotope must be able to withstand dilution, so that the radioactivity of the final sample can not be lower than the background, generally speaking, radioisotopes are not completely uniformly diluted in the organism, may be in some organs, tissues, cells, some molecules have selective accumulation, the part of the accumulated radioactivity will be very strong, this kind of radioactivity will be very strong. In this case, the tracer dosage should be considered in terms of the accumulation rate of the tracer in the relevant part. In cell culture, slice holding, enzyme reaction and other tracer experiments, should be based on the purpose of the experiment, reaction time and reaction volume to consider the amount of tracer, usually less than a microcurie or a few microcurie. Because of the radiation effect of radioisotopes, should be used according to the type of radionuclide, the amount of control in the maximum permissible dose (maximun permissible dose), so as to avoid the radiation effect caused by the dose is too large, to the experiment to bring large errors.
2. Selection of tracer delivery route
The overall tracer experiment, according to the purpose of the experiment, should be easy to absorb, easy to operate the delivery route, generally given the number of small volume, the requirement to give the dose accurately, to prevent possible loss and unnecessary pollution. In vitro tracer experiments, should be based on the experimental design of the experimental steps of a certain link to add a certain dose of tracer to the reaction system, and strive to operate accurately and carefully.
3. Preparation of radioactive biological samples
According to the purpose of the experiment and the tracer labeled radioisotope nature of the preparation of radioactive biological samples, where the nature of the radioisotope is the form of biological sample preparation is the main basis. If it is a tracer that releases r rays, the sample preparation is relatively easy, as long as the quantitative removal of the measured material into the well NaI (TL) crystal can be measured; if it is a tracer that releases hard β-rays, it is necessary to make the biological samples into a thin thickness of the liquid, or the liquid laying samples and then dried, but also can be grayed out after the laying of samples, put into plastic crystal scintillator to determine, or the use of bell-type Gaiyi Ge counting tubes to detect; if the labeled If the labeled isotope emits only soft beta rays, then the sample should be made into a liquid scintillation sample (see chapter "Radioactivity Measurements" for details) and measured in a liquid scintillation counter. Regardless of the measurement method used, the sample should be quantitatively collected. For some radioactively dispersed samples, the sample should be concentrated appropriately, e.g. to determine the radioactivity of proteins in tissues, the proteins should be extracted and then prepared as a corresponding measurement sample. Some samples need to use ashing, but ashing is not suitable for volatile isotopes or volatile tissue samples.
4. Measurement of radioactive samples
Measurement methods are categorized into absolute and relative measurements. Absolute measurement is to measure the actual radioactive intensity of the sample to find out the actual decay rate of the labeled isotopes in the sample, in the absolute measurement, to correct for the effect of some factors on the measurement results, these factors include the relative stereo angle of the instrument probe for the radioactive source, the probability of counting the rays received by the probe, the backscattering, self-absorption of the radioactive source, and so on. The relative measurement is only a relative measurement of the intensity of radioactivity on a fixed probe, without pursuing its actual decay rate. In general tracer experiments, most of the relative measurements are used to compare the differences between samples. During relative measurements, care is taken to keep the geometric position between the sample and the detector fixed. The effect of geometric conditions is the most important influence in radioactivity measurements. When two samples with the same radioactivity intensity are placed in different geometric positions in the measurement, or the geometric conditions caused by the sample preparation process are different, their counts will be much different, especially when the distance between the sample and the probe is relatively close, the difference between the two count rates will be very large. However, when the sample is farther away from the probe, the difference in count rates is significantly reduced due to the smaller relative steric angle formed between the sample and the probe. When measuring the radioactivity intensity of 3H markers by the paper sheet method, it is necessary to pay attention to the position of the paper sheet in the scintillation vial, a batch of samples should be the same, and if the filter paper is cut into a circle as a support, it is best for the diameter of the circle to be equal to the diameter of the bottom of the scintillation vial, so as to ensure that the filter paper is fixed in the position of the scintillation vial. Reduce the impact of geometric conditions on the measurement of radioactivity can start from three aspects: (1) the choice of large detection window detector, such as photomultiplier tubes for the probe detector; (2) in the sample preparation, pay attention to try to make the sample into a point source, so that when the sample of the radioactive intensity of the weak, due to the distance from the detection window is closer to the possibility of causing the impact of horizontal displacement can be ignored; (3) regardless of the sample distance from the window of the probe is far from the sample should be placed in the vertical axis of the window. should be placed on the vertical axis of the detection window to reduce the relative stereo angle between the sample and the detection window.
(C) radioactive decontamination and radioactive waste disposal
Radioactive experiments, whether it is the end of each experiment or phase of the experiment, there may be varying degrees of radioactive contamination and the emergence of radioactive waste, therefore, after the end of the experiment, decontamination treatment and radioactive waste disposal. If necessary, in the course of the experiment, it is necessary to make the decontamination and clean up the work of radioactive waste.
Three, isotope tracer method in biochemistry and molecular biology
Radioisotope tracer method in the field of biochemistry and molecular biology is very widely used, which reveals the secrets of the physical and chemical processes in the body and the cell, and elucidates the material basis of life activities plays an extremely important role. In recent years, there have been many new developments in isotope tracer technology on the basis of the original, such as double-labeling and multi-labeling technology, stable isotope tracer technology, activation analysis, electron microscope technology, isotope technology combined with other new technologies, etc. As a result of these developments, biochemistry and molecular biology have become more and more important. As a result of the development of these techniques, biochemistry from static into dynamic, from the cellular level into the molecular level, elucidated a series of major issues, such as the genetic code, cell membrane receptors, RNA-DNA reverse transcription, etc., so that mankind's understanding of the basic phenomena of life opens up a new way. The following is only an introduction to several major aspects of the application of isotope tracer technology in biochemistry and molecular biology.
1. The study of substance generation and release of Xie
There are many kinds of substances in the body, exactly how they are transformed, if the appropriate isotope markers are applied as tracers in the study to analyze the change of isotope content of these substances, it can be known that they are transformed into each other, and it can also be differentiated who is the predecessor, who is the product , analyze the isotope tracers are present on which atoms of the substance molecules, and analyze the isotope tracers are present on which atoms of the substance molecules. Analyzing which atoms of a substance molecule the isotope tracer is present on can further deduce the transformation mechanism between various substances. In order to study the biosynthesis of cholesterol and its metabolism, the method of labeling precursors was used to reveal the pathway and steps of cholesterol generation, and it was experimentally proved that all compounds that can be transformed into acetyl coenzyme A in the body can be used as raw materials for the generation of cholesterol, and that the entire biosynthesis process from acetic acid to cholesterol consists of at least 36 steps of chemical reactions, and that there are twenty intermediates between squalene and cholesterol, and that there are no intermediates between squalene and cholesterol. The biosynthetic pathway of cholesterol can be simplified as follows: acetic acid → methyl dihydroxyvaleric acid → cholesterol Another example is that in the study of the source of cholesterol in the liver, the tracer experiments using the radioisotope marker 3H-cholesterol as an intravenous injection show that most of the radioactivity enters the liver and then reappears in the feces, and thyroxine accelerates the process, which can be used to show that the liver is the main organ that processes plasma cholesterol, and that the thyroid gland can accelerate this process. The mechanism by which the thyroid gland lowers blood cholesterol levels lies in its accelerating effect on the transfer of plasma cholesterol to the liver.
2. Research on the transformation of substances
The law of mutual transformation of substances in the organism is an important essential content of life activities. In the past, the research on the transformation of substances has generally adopted the method of in vitro enzymology, but the results of in vitro enzymology are not necessarily representative of the overall situation, and the application of isotope tracer technology has greatly shortened the period of experiments related to the transformation of substances, and has made it possible for the study to be conducted in an isolated, holistic and non-invasive manner. Moreover, it can be applied in the case of isolated, whole and cell-free system, and the operation is simplified, the sensitivity of determination is improved, and not only the qualitative, but also the quantitative analysis can be done. In the elucidation of the conversion of ribonucleotides to deoxyribonucleotides, the double labeling method is used, and the product is measured by double labeling or its radioactivity is measured separately after chemical separation. For example, in the guanine nucleotide (GMP) base and ribose were labeled with 14C, in the isolated system so that the reference to deoxyguanine nucleotide (dGMP), and then the original marker and the product (double labeled GMP doped dGMP) were acid hydrolysis and chromatographic separation, respectively, to determine the radioactivity of their respective bases and pentose sugar, and found that the ratio of the radioactivity of the two parts of them is basically This proves that the pentose of the product dGMP is the pentose of the original labeled GMP, and there is no other source, otherwise the ratio of the base and ribose of the product dGMP must be significantly different from the ratio of the two parts of the original labeled GMP. This experiment shows that the deoxygenation of pentose is carried out in the case that the base and pentose are not separated from each other, thus proving that deoxyribonucleotides are directly converted from ribonucleotides, and not that ribonucleotides are first broken down into ribose and base, and that the base is then rewired to the deoxyhanging ribose. Cell-free tracer experiments can analyze the conditions of the transformation of substances in the cell, such as 3H-dTTP as the precursor for DNA doping tracer experiments, according to a certain experimental design after doping, the determination of the product DNA radioactivity, as a newly synthesized DNA detection index.
3. Dynamic equilibrium research
Elucidating that substances in living organisms are in a constantly renewed dynamic equilibrium is one of the major contributions of radioisotope tracing method to life sciences. Introducing appropriate isotope markers into the body, and determining the change of isotope content in substances at different times, we can understand the change of the substances in the body, quantitatively calculate the metabolism rate of substances, and calculate the update rate of substances in the body. The metabolic rate of a substance in the body can be calculated quantitatively, and the rate and time of renewal of a substance can be calculated. Various substances in the body have different sizes of metabolic reservoirs, and the size of metabolic reservoirs can be calculated by isotope dilution method.
4. Analysis of trace substances in biological samples
Before the application of radioisotope tracer technology, due to the loss of sample preparation, resulting in a low recovery rate and measurement of sensitivity is not high, making the normal function of the body plays a very important role in the determination of trace substances is not easy. In recent years, the rapid development and wider application of radioimmunoassay (radioimmunoassay) technology is an ultra-micro-analysis method, which can determine more than 300 kinds of substances, including hormones, including steroid hormones, peptide hormones, non-peptide hormones, protein substances, cyclic nucleotides, enzymes, tumor-related antigens, antibodies, as well as pathogens, micro-drugs and other substances. Other substances.
5. Nearest neighbor-sequence analysis method
Radioisotope tracer technology is one of the most important means of molecular biology research, and it plays a great role in protein biosynthesis research from DNA replication, RNA transcription to protein translation. The role of radioisotope tracing technique is very important in molecular biology research. Recently, the neighboring sequence analysis method applied the isotope tracer technology combined with the enzyme cutting theory and statistical theory, the research confirms the base arrangement law in the DNA molecule, in vitro for the synthesis of DNA experiments: carried out in four batches, each batch of deoxyribonucleoside triphosphate labeled with a different kind of 32P, 32P labeled in the position of the pentose 5'C. After synthesizing under complete conditions, the original bases were opened with specific enzymes to make the original base 5'C-P bond. -P bond, so that the 32P attached to the original base through the pentose 5'C is moved to the 3'C of the nearest neighboring another mononucleotide . The molecular biological basis of DNA replication and RNA transcription was first proposed by nearest neighbor sequence analysis, which led to the establishment of molecular hybridization techniques, such as using phage T2-DNA as a template to make [32P]RNA, taking a certain amount of T2-DNA and some other DNA to add to this [32P]RNA, heating to make the DNA double-stranded open, and temperature incubation, and then centrifugation with a density gradient or a microporous The DNA-[32P]RNA complex was separated by density gradient centrifugation or microporous membrane to measure its radioactivity, and the experimental results showed that only the DNA of bacteriophage T2 could form a radioactive complex with this [32P]RNA
. Thus proved that RNA and DNA template bases are special pairing complementary relationship, with molecular hybridization technology also confirmed the reverse transcription phenomenon from RNA to DNA. In addition, the contribution of radioisotope tracer technology to molecular biology is also manifested in: (1) the protein synthesis process of the three successive phases, that is, the peptide chain initiation, extension and termination of the study; (2) the isolation and purification of nucleic acids; (3) nucleic acid terminal nucleotide analysis, sequence determination; (4) nucleic acid structure and function of the relationship between the (5) RNA in the genetic information of the how the nucleotides in the arrangement of the order of the transmission of the amino acids in the egg mass to the Research and so on. In order to better use of radioisotope tracer technology, in addition to relying on the high quality of the tracer and the high sensitivity of the nuclear detector, the key also lies in the scientific basis of the concept and creative experimental design and a variety of new technologies integrated application.