1 RFLP
This technology was founded by Grodzicker in 1974. After the genome DNA of a specific biological type is completely digested by a restriction enzyme, homologous allele fragments with different molecular weights will be produced, or the basic principle of RFLP labeling technology is to detect the mutations of these fragments through electrophoretic separation. For example, point mutation (the new generation and removal of restriction sites) and recombination of a piece of DNA (such as the change of length between restriction sites caused by insertion and deletion) can lead to the change of restriction alleles, thus producing RFLP. The technology includes the following basic steps: DNA extraction; Digestion with DNA restriction enzyme; Separation of restriction fragments by gel electrophoresis; Transfer these fragments to the easy-to-operate filter membrane according to the original order and position; Using DNA labeled with radioactive isotopes or non-radioactive substances as probes to hybridize with DNA on the membrane (called Southern hybridization); Autoradiography or enzyme detection showed that different materials were polymorphic in the restriction fragment of the probe.
The main characteristics of RFLP markers are: (1) distributed in the whole genome, and the number is almost infinite; (2) There is no phenotypic effect, and it is not limited by developmental stage and organ specificity; (3)*** dominance, which can distinguish homozygotes from heterozygotes; (4) The results are stable and reliable; (5)DNA needs a lot, and the detection technology is complex, so it is difficult to be used in large-scale breeding practice.
2 RAPD
Founded by Williams = 1990, its basic principle is consistent with PCR technology.
PCR is a method for rapid amplification of specific genes or DNA sequences in vitro. Mullis was the first to establish a reaction system in a test tube. After a few hours, a very small number of target genes or specific DNA fragments can be amplified millions of times. Its principle is similar to the DNA replication process in cells. First, when heated at a temperature close to the boiling point, a double-stranded DNA molecule is separated into two single-stranded DNA molecules. Then, using single-stranded DNA as a template, DNA polymerase uses four deoxynucleoside triphosphates (dNTPs) in the reaction mixture to synthesize new complementary DNA strands. The above process is a cycle, and the product of each cycle can be used as a template for the next cycle. After 20-30 cycles, the specific DNA fragments between the two primers can be copied in a large number with geometric numbers.
RAPD marker technology is to randomly amplify genomic DNA with one (sometimes two) random primers (usually 8- 10 bases), and then separate the amplified genomic DNA of genetic material by gel electrophoresis. If DNA fragment insertion deletion or base mutation occurs in a specific primer binding region, the distribution of primer binding sites may change accordingly, resulting in the increase or deletion of PCR products or the change of molecular weight. If PCR products are increased or lacking, RAPD markers will be produced.
The main characteristics of RAPD markers are: (1) No DNA probe and no sequence information are needed to design primers; (2) Dominant inheritance (very few * * * dominance), which can not distinguish heterozygotes from homozygotes; (3) The technique is simple and does not involve molecular hybridization and autoradiography; (4) The demand for DNA samples is small, the price of primers is cheap and the cost is low; (5) The repeatability of the experiment is poor and the reliability of the results is low.
3 AFLP
The AFLP marker invented by Zabeau and Vos in 1993 is the polymorphism of the amplification product produced by selective amplification of the restriction fragment of genomic DNA, which essentially shows the length polymorphism of the restriction fragment of restriction endonuclease, but this polymorphism is detected by the difference of the length of the amplification fragment. This technology combines the stability of RFLP with the simplicity and efficiency of PCR technology. At the same time, it can overcome the shortcomings of instability of RFLP band and RAPD technology. The basic technical principle and operation steps are as follows: firstly, genome DNA is digested with restriction endonuclease to form many random restriction fragments with different sizes; Then the two ends of these fragments are connected with specific oligonucleotide linkers; Then design primers according to the linker sequence. Because there are too many restriction fragments, it is difficult to separate all the amplified products on the gel. Therefore, 1-3 selective bases are added to the third end of the primer, so that only those fragments that can be paired with selective bases can be combined with the primer and amplified as templates, thus achieving the purpose of selectively amplifying restriction fragments. Finally, these specific amplification products were separated by polyacrylamide gel electrophoresis.
The main characteristics of AFLP markers are as follows: (1) Because there are many restriction endonucleases and selective base types that can be used in AFLP analysis, the number of markers produced by this technique is infinite; (2) In typical AFLP analysis, the bands of each reaction product are between 50- 100, so multiple loci can be detected simultaneously in one analysis, and the polymorphism is extremely high; (3) * * dominance, typical Mendelian inheritance; (4) High resolution and reliable results; (5) At present, this technology is protected by patent, and the analysis kit is expensive and the experimental conditions are harsh.
Four-time SSR(SSLP)
SSR (microsatellite DNA), discovered by Moore in 199 1 year, is a kind of DNA sequence consisting of several bases (mostly 1-5), which is generally short in length and widely distributed in different parts of the genome, such as (CA) N (AT) N (GGC). This leads to the high variability of SSR length, which is the basis of SSR markers. Although microsatellite DNA is distributed in different positions in the whole genome, its two ends are mostly conservative single-copy sequences, so we can design a pair of specific primers according to the sequences at these two ends, amplify the core microsatellite DNA sequence between them by PCR technology, and obtain its length polymorphism, that is, SSR markers, by electrophoresis analysis technology.
The main characteristics of SSR markers are: (1) abundant and widely distributed in the whole genome; (2) There are many allelic variations; (3)*** dominant marker, which can distinguish heterozygote from homozygote; (4) The experiment has good repeatability and reliable results; (5) Because it is necessary to know the sequence information at both ends of the repeated sequence when creating a new tag, it is difficult to develop and the cost is high.
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