1. Extract the DNA contained in various biological samples such as hair, blood stains, sperm spots, human tissues or bones.
2. The restriction endonuclease paired with the probe was selected and cut at the position of long-chain DNA, so that the long-chain DNA with large molecular weight was cut into many small fragments with different lengths.
3. In a gel electrophoresis apparatus with a long rubber plate, the DNA fragments after complete enzymolysis are electrophoresed, and each fragment after enzymolysis will be separated according to its length in an electric field.
4. First denature the double-stranded DNA fragments separated from the gel plate into single-stranded DNA fragments with alkaline solution, and then sandwich the gel plate in nylon membrane to make these single-stranded DNA fragments.
The fragment was blotted, transferred and permanently fixed on the nylon membrane.
5. The radioactive DNA probe hybridizes with the single-stranded DNA fragment on the nylon membrane.
6. When the radioactive film is overlapped with the nylon film, the radioactive probe on the nylon film will emit X-rays to expose the film, thus developing DNA fragments with different lengths hybridized with the probe on the film. The banded pattern of this characteristic DNA fragment is called DNA fingerprint.
Attach a little knowledge of popular science of DNA:
Establishment and development of 1 DNA fingerprint
Nearly a hundred years' research holds that any genetic analysis is based on genetic markers, and the value of any genetic marker lies in its variability (that is, polymorphism). The study of genetic polymorphism plays a very important role in promoting the development of anthropology, genetics, immunology and forensic medicine, and clarifying the pathogenesis of some diseases and even assisting diagnosis. However, previous studies mostly used various phenotypes, physiological defects, isoenzymes and polymorphic proteins as genetic markers, and inferred the corresponding genetic genes by indirect analysis.
At the end of 1970s, with the appearance of restriction endonuclease and recombinant DNA technology and the rapid development of molecular biology, the research on genetic markers turned to DNA molecule itself. Because all kinds of genetic information are contained in DNA molecules, the difference between biological individuals is essentially the difference of DNA molecules, so DNA is considered as the most reliable genetic marker. The difference of some DNA sequences can be reflected by the change of restriction fragment length, which is called restriction fragment length polymorphism (RFLP) and is caused by point mutation, DNA rearrangement, insertion or deletion [1]. With the in-depth study of RFLP, highly variable DNA sequence, the most variable sequence in the genome, has been discovered, which makes the development and application of DNA genetic markers leap forward.
In 1980, Wyman and White described the first multi-allele human DNA marker with high polymorphism. Soon, the same hypervariable marker was found in the 5' end region of insulin gene and the 3' end of C-Haras I oncogene. Three other markers were found around the α -globulin gene group [2]. In 1982, Bell et al. [3] confirmed that these highly polymorphic regions are connected in series with repetitive short sequence units, and the difference in the number of repetitive units leads to this high variability. Because of these structural characteristics, people call these regions small satellites or high variable regions or variable number of tandem repeats.
In 1985, Jeffreys et al. [4] used the tandem repeat sequence in the first intron of myoglobin gene as a probe, and screened 8 recombinant clones containing tandem repeat sequence (microsatellite) from human gene library. Sequence analysis showed that the eight microsatellite repeat units were not exactly the same in length and sequence, but all had the same core sequence, namely GGCCAGGA/GGG. They used two small multi-core satellites (Polycore satellites).
-llite)33.6 and 33. 15 probes were used for southern hybridization, and hybridization maps containing more than 10 bands were obtained under low stringency conditions. The positions of bands on the hybridization maps of different individuals are just as different as human fingerprints, which Jeffrey called DNA fingerprints [5], also known as genetic fingerprints.
RFLP DNA fingerprinting technology can not be widely popularized because of its complex method, long period and high experimental conditions. 1990, Williams et al. [6] reported AP-PCR technology for the first time, and Welsh and McCell and [7] also independently carried out this work, thus making DNA fingerprinting technology more widely used. AP-PCR technology uses randomly designed 1 or 2 primers to amplify template DNA. Generally, PCR with 1 ~ 6 cycles is first carried out under low stringency conditions, that is, at high Mg2+ concentration (higher than that of 1.5mmol/L of traditional PCR) and low annealing temperature (36℃ ~ 50℃). The basic principle is that under the condition of low strict renaturation, the primer and the incomplete complementary sequence of template DNA form a mismatch, and the mismatched primer extends along the template chain under the action of DNA polymerase to synthesize a new chain. When another single strand of template DNA also has primer mismatch within a certain distance, the DNA between two mismatched primers can be amplified. However, this mismatch does not happen randomly. There must be a certain complementary sequence between the primer and the template, especially at the 3' end of the primer, which can produce different amplification fragments or combinations. Through DNA fingerprinting technology, differential fragments in paired DNA samples can be obtained for cloning, sequencing, chromosome location and biological function research of gene fragments.
Yang Jianchang and others in China [8] successfully established a brand-new DNA fingerprinting technology by using the principle of PCR, which is called random primer PCR human DNA fingerprinting technology (APHDP). In addition, they also developed application software to process DNA fingerprint data and applied it to personal identification, genetic quality and related characteristics of diseases.
Two kinds of probes used in DNA fingerprinting technology
Since the establishment of DNA fingerprinting technology, it has been widely used in the analysis of evolutionary relationship between animals and plants, genetic relationship and forensic medicine. It is precisely because DNA fingerprinting technology has shown great vitality in nucleic acid analysis that many scholars have done a lot of work around the probes used in this technology. In addition to the probes used by Jeffrey et al. [5], many high-level probes have been produced by artificial chemical synthesis or extraction from biological tissues and then amplification. Up to now, the probes used in DNA fingerprinting technology include probe 33. 15, 33.6 [5], phage MB [9], porcine clone p83, PGB 725, Poly (GT) containing 18. 1, (GTG) 5/. At the same time, the labeling of probes has also made great progress. According to their structures, they can be roughly divided into microsatellite probes and simple sequence repeats, which include microsatellite probes and oligonucleotide probes. The core sequence of microsatellite probe is 33bp, which is often located in the front end region before human autosome, while the microsatellite probe is between 10~20bp, while the oligonucleotide probe is below 10 ~ 20bp, which is widely distributed on the whole human chromosome, or in intergenic regions or introns.
In 1988, Wu Xinyao of China et al. [12] based on the principle that DNA fingerprinting is a repetitive sequence in the human genome and the fact that the homology of human and mouse myelin basic protein (MBP) gene cDNA is higher than 90%, a segment of the 3' end of mouse MBP CDN (a highly repetitive sequence in the non-expression region) is selected to be similar to this kind of repetitive sequence in the human genome. Using the fragment with the length of 0.8 1kb as a probe, the restriction fragment of human DNA digested by Hae was detected. There are 22 bands in the population, and no two bands in 30 unrelated individuals are exactly the same, which shows that this method has high individual specificity. This is the first time that China has found a probe for DNA fingerprint through its own efforts.
3. Application of DNA fingerprinting technology 3. 1 Compared with the previous blood group identification methods, DNA fingerprinting technology has unparalleled advantages in the field of forensic medicine. It has become a tool for identifying crimes, paternity testing and determining the genetic relationship between individuals [5 13]. Subsequently, domestic scholars Li Boling [14], Jiang Xianhua [15], Wu Xinyao and others [12] also studied this technology one after another, and applied it to the identification of actual cases, which solved difficult cases that could not be solved in the past, such as trace blood and personal identification of some corrupt fragments.
3.2 Application in Animal and Plant Science
3.2. 1 The study of biological demography can estimate linkage disequilibrium, compare allele frequencies and estimate recombination rate between different individuals, which is helpful to establish the position and relationship of an individual in the population, especially in the study of fungal population. Many fungi can reproduce sexually and asexually, but it is not clear when, how and how much to reproduce. Using DNA fingerprinting technology, sexual offspring and asexual offspring can be distinguished and the natural distribution of fungi in a certain area can be determined [1, 16].
3.2.2 Determination of genetic distance between species, species classification and identification Jeffreys et al. [5] think that tandem repeats (VNTR) with high copy number among different members of a population are particularly suitable as polymorphic markers in genetic analysis, and the instability of simple sequence repeat can lead to rapid changes in the length of VNTR. According to the frequency of VNTR segregation and recombination in a family or breeding population, the genetic distance can be determined. The genetic relationship among individuals can be determined by the statistical formula: D=2Nab/(Na+Nb). The greater the value of d, the closer the genetic relationship and the smaller the genetic distance. The smaller the D value, the farther the genetic relationship and the greater the genetic distance. Therefore, DNA fingerprinting technology can be used to detect the genetic relationship between different species, the same species and different individuals of the same species, and can also be used to determine the parents of hybrid offspring, separate the population of hybrid offspring, detect the polymorphism of near-isogenic lines (or similar lines) and locate the detected genes. Welsh et al. [7] analyzed the DNA fingerprint of Borrelia brucei strain and found that the pathogen of Lyme disease is actually composed of three different populations. Luo Chaoquan and others [12] used AP-PCR to identify Toxoplasma gondii strains, which pioneered the biological classification by DNA fingerprinting technology in China.
3.3 Application in epidemiology Because DNA fingerprinting has the following characteristics: ① It can reflect the variability of genome; ② High variability; ③ Simple and stable inheritance; ④DNA fingerprinting has somatic stability. Therefore, compared with general epidemiological methods, it has incomparable advantages and is an effective tool for epidemiological investigation. Jan DA et al. [17], Denise Chevrel-Dellagi et al. [18] used IS6 1 10 sequence as a probe to analyze the DNA fingerprints of Mycobacterium tuberculosis strains, investigate the types of tuberculosis in the world, analyze the epidemic situation and improve the methods of controlling tuberculosis. Yang Zhenhua et al. [19] isolated mycobacterium tuberculosis strains from 67 patients for DNA fingerprinting analysis, and found that it is easy to identify epidemic links when PTBN 12 was isolated, thus providing strong evidence for rapid disease control. In China, Tong Xiaomei et al. [20] used random amplified polymorphic DNA fingerprinting technology to analyze the pathogens of 14 newborns with hospital infection, and found that the DNA fingerprints of Staphylococcus warner strains carried in children were completely consistent with those carried by medical staff, thus proving that the pathogen of this infection was Staphylococcus warner, and the source of infection was medical staff carrying this pathogen. Guo Yongjian et al. [2 1] conducted RAPD fingerprint analysis and serological typing on 30 cases of 3 1 strain of Pseudomonas aeruginosa detected in 12 1 obstetric newborns within 6 months. The results showed that Pseudomonas aeruginosa broke out in obstetric newborns, and the 0∶6/R∶ 1 type was an explosive epidemic strain.
3.4 Diagnosis and treatment of diseases In view of the above characteristics of DNA fingerprints, DNA fingerprints are widely used in the diagnosis and treatment of some diseases. Morral [22] and others found that the flank of exon 9 of CF gene contains a small satellite region, and the allele 2.6 band is often linked with △F508, with the association rates of 50.6% and 4 1.6%. △F508 is the most important pathogenic mutation. If only 2.6 allele is found in the electrophoretogram of suspected patients, the disease can be preliminarily diagnosed. At present, high microsatellite regions have been found in or near Wilson's disease, peripheral neurofibroma, adult multiple kidneys, dopaminergic dystonia, Frecbrech * * ataxia, Kallmunm syndrome, retinopathy and other genes, so that gene diagnosis can be carried out. Okamoto R [23] used DNA fingerprinting technology to predict the recurrence of chronic myeloid leukemia after bone marrow transplantation, and achieved success.
3.5 Research on tumor Tumor is a multi-factor and multi-stage change process. The reasons are complicated and varied, but in the final analysis, it is the change of DNA. Generally speaking, the DNA fingerprints of cancer tissues and metastases are different from those of normal tissues or peripheral blood cells. It is common that one or more bands are missing, the density of one or more bands is reduced, or new bands appear in cancer tissues. Thein et al. [24] studied the changes of DNA fingerprints of patients with gastrointestinal tumors with 33.6 and 33. 15 as probes, and found that the DNA fingerprints of cancer tissues of patients with gastrointestinal tumors all changed, and thought that somatic mutation was species-specific. Liu Shuang et al. [25] used RAPD (Random Amplified Polymorphic DNA) analysis technology to analyze the cancer tissues and non-cancer tissues of 6 patients with liver cancer, and found that the RAPD fingerprints of genomic DNA of all liver cancer tissues were different, among which 3 matched liver cancer genomes all had the same randomly amplified fragment of 0.9Kb. Yang Jianchang et al. [8] used APHDFF technology to detect the blood DNA fingerprints of 28 patients with nasopharyngeal carcinoma and found 3 DNAs. Wang Dai et al [26] used LE 1 1.8 and Mb probes to detect the gene rearrangement of peripheral blood or bone marrow cells in 12 children with acute myeloid leukemia by Southern hybridization. The results showed that the initial or recurrent DNA fingerprints increased or decreased compared with the complete remission, so it was considered that there was gene rearrangement in leukemia cells of children with acute myeloid leukemia.
References:
/question/ 13 180448 . html
Respondent: xy _ vanilla- Juren Level 4 8-1113: 31.
Let me comment >>
The questioner's evaluation of the answer: too much, my friend! I asked, but you haven't fully answered. Thank you all the same! The evaluation has been closed, and there are currently 3 evaluations.
okay
100% (3) is not good
0% (0)
Related content
Application of DNA fingerprinting technology and related cases
About the Olympics
How many "identity cards" does the human body have?
DNA fingerprinting technology uses enzymes to cut into fragments. What enzyme is used?
Identical twins should have the same DNA, and if they die, they can pass. ...
The same point of view: technology application fingerprint principle
Other answers *** 3
DNA fingerprinting is a new technology in molecular biology. It is an important means to distinguish the differences between different kinds of organisms and between the same species at the molecular level. It has very important applications in forensic identification, species origin and evolution research, establishment of DNA fingerprint database of animals, plants and microorganisms, animal husbandry research, cancer research, disease diagnosis, paternity test and so on. This paper briefly expounds the research progress and application of DNA fingerprinting technology.
Responder: Shui Xin Yujun-Trainee Magician Level 2 8- 1 1 13:33.
Dna fingerprinting refers to a completely individual-specific dna polymorphism, and its individual recognition ability is enough to match a hand.
Fingerprints, hence the name. It can be used for personal identification and paternity test.
Identification of DNA fingerprints
________________________________________
1984, geneticist Jefferys of the University of Leicester and his collaborators used the isolated human microsatellite DNA as a gene probe for the first time to hybridize with the digested fragment of human nuclear DNA, and obtained a hybridization band pattern with different lengths composed of alleles of multiple loci. This pattern is rarely identical to two people, so it is called "DNA fingerprint", which means it is unique to everyone like human fingerprints. The image of DNA fingerprint shows a series of stripes on X-ray film, much like bar codes on commodities. DNA fingerprinting technology has created a variety of means to detect DNA polymorphism (different individuals or different populations have different DNA structures), such as RFLP (Restriction endonuclease fragment length polymorphism) analysis, tandem repeat sequence analysis and RAPD (Random Amplified Polymorphic DNA) analysis. All kinds of analysis methods are based on the polymorphism of DNA, resulting in DNA fingerprints with high individual specificity. DNA fingerprint is the most attractive genetic marker because of its high variability, stable heredity and simple Mendelian inheritance.
DNA fingerprints have the following characteristics: 1. High specificity: the research shows that the probability of two random individuals having the same DNA pattern is only 3×10-11; If two probes are used for comparison at the same time, the probability of two individuals being the same is less than 5× 10- 19. The world population is about 5 billion, that is, 5× 109. Therefore, unless they are identical twins, it is almost impossible for two people to have the same DNA fingerprint. 2. Stable inheritance: DNA is the genetic material of human beings, and its characteristics are inherited by parents. It is found that almost every band in DNA fingerprint can be found in the atlas of one of its parents, which conforms to the classical Mendelian inheritance law, that is, 50% of the characteristics of both sides are passed on to future generations on average. 3. Somatic cell stability: that is, the DNA fingerprints produced by different tissues such as blood, muscle, hair and semen of the same person are completely consistent.
1985, Dr. Jefferys first applied DNA fingerprinting technology to forensic identification. 1989 this technology was approved by the us congress as a formal means of court material evidence. China police solved thousands of difficult cases by using DNA fingerprinting technology. DNA fingerprinting technology has many advantages that traditional forensic examination methods do not have. For example, it can still extract DNA from semen spots and blood samples four years ago for analysis. If mitochondrial DNA is used for examination, the time will be prolonged. In addition, DNA fingerprinting technology was used to identify the found Millennium corpse, the remains of Tsar Nicholas, who was executed during the Russian revolution, and the remains of the late US Secretary of Commerce Brown and his entourage who died in an accident in the former Yugoslavia not long ago.
In addition, it is used in human medicine to identify individuals, determine kinship, medical diagnosis and find genetic markers related to diseases; Can be used for the origin and evolution of animal populations in animal evolution; In species classification, it can be used to distinguish different species, and also has the potential to distinguish different strains of the same species. It is also widely used in crop gene mapping and breeding.
The basic operation of DNA fingerprinting is to extract DNA from biological samples (DNA is generally partially degraded) and highly mutated sites (such as VNTR system and tandem repeat microsatellite DNA). ) or amplify the complete genomic DNA by PCR technology, then cut the amplified DNA into DNA fragments, separate them by molecular weight by agarose gel electrophoresis, transfer them to nylon filter membrane, and then hybridize the labeled microsatellite DNA probe with DNA fragments with complementary base sequences on the membrane.
Agarose gel electrophoresis is a routine method to separate, identify and purify DNA fragments. The location of DNA in the gel can be determined by staining with ethidium bromide, a low concentration fluorescent embedding dye. If necessary, DNA bands can also be recovered from the gel for various cloning operations. The resolution of agarose gel is lower than that of polyacrylamide gel, but its separation range is wider. DNA with a length of 200bp to nearly 50kbp can be separated by agarose gel with various concentrations. DNA with a length of 100kb or more can be separated by pulsed electric field gel electrophoresis with periodically changing electric field direction.
In the routine operation of genetic engineering, agarose gel electrophoresis is the most widely used. It usually uses a horizontal electrophoresis device to conduct electrophoresis under an electric field with constant intensity and direction. DNA molecules are negatively charged in gel buffer (usually alkaline) and migrate from negative electrode to positive electrode in electric field. The mobility of DNA molecules is influenced by molecular size and conformation. The influence of electric field intensity and direction, base composition, temperature and embedded dyes.