Restrictive fragment length polymorphism

Biological terminology

Restriction fragment length polymorphism (RF LP) is referred to as PCR-RFLP analysis. It is mainly to design appropriate amplification primers so that the amplified fragment includes one or more polymorphic restriction enzyme recognition sequences. After PCR amplification, the PCR product is cut with the restriction enzyme, and VNTR is digested according to Amp FLP STR and other repeat sequences are highly polymorphic due to the number of repeat units. Therefore, specific primers on both sides of the repeat sequence were used for PCR amplification, and the amplified fragments were highly polymorphic. These allelic fragments with different lengths could be separated by PAGE.^[1]

Restriction fragment length polymorphism (abbreviation RFLP ）The principle of technology is detection DNA stay Restriction endonuclease Enzyme digestion The size of the specific DNA fragment formed after. Therefore, any Restriction site Mutations such as point mutation (New generation and removal of enzyme digestion sites) and a segment of DNA organization (For example, insertion and deletion cause changes in the length between enzyme digestion sites) can lead to the production of RFLP.

Chinese name: Restrictive fragment length polymorphism
Foreign name: RFLP，Restriction Fragment Length Polymorphism

Proposed time: 1980
Proposer: Bostein
Role: First generation DNA Molecular marker technology

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Technical Introduction

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Restrictive fragment length polymorphism（ RFLP , Restriction Fragment Length Polymerism) RFLP technology was proposed by human geneticist Bostein in 1980. It's the first generation of DNA Molecular marker technology 。 Donis Keller used this technology to build the first human genetic map 。 DNA molecule Horizontal Polymorphism Detection technology is the basis of genome research. RFLP(Restriction Fragment Length Polymorphism, Restriction fragment length polymorphism ）Has been widely used genome Genetic map construction Gene mapping as well as Biological evolution And classification. RFLP is based on the genomes of different varieties (individuals) Restriction endonuclease Of Restriction site Base Mutation, or base insertion or deletion between enzyme digestion sites, resulting in Enzyme digestion The fragment size has changed. This change can be detected by hybridization with specific probes, so that the difference in DNA level (i.e. polymorphism) of different varieties (individuals) can be compared. The comparison of multiple probes can establish the evolution and classification relationship of organisms. The probe used is from the same or different species Genomic DNA The clone of can be used as a Molecular marker (Mark), to construct the molecular map.

When a trait (gene) and a molecular marker (s) are separated together, it indicates that the trait (gene) and Molecular marker linkage 。 Between molecular markers and traits Exchange value The size of, that is, the distance between the target gene and the molecular marker, so that Gene mapping On the molecular map. Molecular markers are cloned on plasmids and can be reproduced and preserved. After different restriction endonucleases cut genomic DNA, the types of fragments cut are different. Therefore, restriction endonucleases and molecular markers form different combinations for research. The commonly used restriction endonucleases are Hind Ⅲ, BamH Ⅰ, EcoR Ⅰ, EcoRV, Xba Ⅰ, and there are several or even thousands of molecular markers. The more molecular markers, the more saturated the constructed map. One of the main objectives of RFLP research is to construct a saturation map.

principle

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This technology is able to recognize DNA molecule And cut the DNA molecule at the specific sequence, that is, to produce the characteristic of restrictive fragments. For individual organisms of different populations, their DNA sequence There are differences. If this difference happens Endonuclease Of Restriction site And make the endonuclease recognition sequence become an unrecognized sequence or this difference makes the DNA sequence that is not an endonuclease recognition site become an endonuclease recognition site. This led to the use of restriction enzyme Enzyme digestion When the DNA sequence is sequenced, one or more restriction sites will be missing, resulting in one or more restriction fragments missing. Thus, when the same restriction endonuclease is used to cut the DNA sequence of different species, it will produce Restriction fragment 。 These fragments are then electrophoretic, transmembrane, denatured, hybridized with labeled probes, washed, and analyzed Polymorphism result.

classification

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Restrictive fragment length polymorphism

DNA structure There are considerable differences among different kinds of organisms. With the gene With the deepening of understanding, it is found that among different individuals of the same organism, although the structure and function of their protein products are identical or only slightly different, there are differences at the DNA level, especially in regions that do not code proteins and regions that do not have important regulatory functions. Most mutations in the DNA sequence are Neutral mutation Which does not affect organisms phenotype Therefore, in the past, little attention was paid to these mutations and traditional genetic methods could not be used to study them. But with Molecular Biology Technology With the continuous development of DNA, it is possible to analyze the mutation of organisms directly from the DNA level. If one of the DNA sequences Base A mutation occurs, causing the DNA sequence of the mutation site to produce (or lose) a certain restriction enzyme site. In this way, when the restriction endonuclease is used to digest this DNA, a restriction fragment different from normal will be generated. In this way, different length restriction fragment types, namely restriction fragments, will appear in different individuals of the same organism Polymorphism （Restriction Fragment Length Polymorphism ，RFLP ）。

RFLP Type

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One is due to a single restriction enzyme site Base This restriction site is lost or acquired due to mutation Polymorphism , so it is called point polymorphism. This kind of polymorphism is actually bimodal, that is, with (+) or without (-). The other is due to large sequence changes within DNA molecules. This type of polymorphism can be divided into two categories: the first is caused by mutations in the DNA sequence, such as deletion, duplication, and insertion. The second is the so-called“ Hypervariable region ”。 Highly variable region is composed of multiple tandem repeat sequences copy number The difference is very large, so the length of hypervariable region changes greatly, which makes the fixed position of restriction endonuclease recognition sites on both sides of hypervariable region move relatively with the size of hypervariable region. Therefore, this type of RFLP is produced due to the different copy numbers of tandem repeats in hypervariable regions, and its prominent feature is the restriction enzyme recognition site itself Base Nothing has changed. What has changed is that it genome The relative position in the. In fact, there are a large number of single base substitutions in the DNA sequence, but the commonly used technology can only detect mutations that affect the recognition site of restriction enzyme.

Haplotype

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different Polymorphism The frequency of tangency (+) in a specific population is different. For example, in a piece of DNA, the frequency of breakpoint A is 0.6 (that is, 60% of people have this breakpoint, while the other 40% do not have this breakpoint at the same site); The frequency of tangent B is 0.4. If these two polymorphic tangents (A, B) are randomly related, then the probability of the simultaneous existence of A and B (++) is equal to the product of the frequency of the existence of each locus, that is, 0.6 × 0.4=0.24, that is, 24% of people have both A and B tangents. If they are non random correlation, then the possibility of simultaneous existence will differ greatly from the expected frequency. This related non Randomness be called Interlocking imbalance 。 If the number of polymorphic breakpoints is n, there can be N power of 2 There are different combinations, and each combination is called one Haplotype (haplotype), the expected frequency of random correlation of each haplotype is the product of the frequency of each locus. However, practice has proved that Polymorphism There is no random correlation between tangents. For example, in β Globin gene There are 8 polymorphic breakpoints from 2 to 9 in the cluster. Theoretically, there should be 256 combinations, namely 256 haplotypes. In fact, there are only three haplotypes. Among them, the β A chromosome (the chromosome carrying the normal β globin gene) accounts for 94% in Greek, Italian and Asian Indian people, while some theoretical combinations do not exist in practice. In theory, the frequency of haplotype (from the tangent point 2 to 9) is 0.46 × 0.48 × 0.7 × 0.27 × 0.83 × 0.52 × 0.37 × 0.32=0.0021, while in fact, the frequency of this haplotype is 0.64, which is very different between the two. This indicates that the polymorphic breakpoints are non random（ Interlocking imbalance ）。 However, there are exceptions, such as tangency 10 (HinfI tangency) and β gene cluster All tangents are Interlocking balance Of. from Haplotype The research on the relationship between β - thalassemia gene and disease gene has found that a β - thalassemia gene has a strong correlation with some haplotypes in a certain population, and there are only a few exceptions. In this way, in this population, as long as we determine the haplotype of a person, we can basically determine whether the person contains a certain mutant gene.

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However, even among people of the same race or region, this linkage is still incomplete. This is mainly reflected in two aspects: first, a certain RFLP Haplotype It is linked to a mutant gene, but the same haplotype can also be found in normal people. Therefore, the application of haplotype cannot say for sure whether a certain individual has the mutant gene. However, even among people of the same race or in the same region, this linkage is not the same. Therefore, the application of haplotype cannot say for sure whether a certain individual has the mutant gene. Second, the same mutation can also be associated with several haplotypes. For these two cases, in order to prenatal diagnosis The haplotype of the fetus can be detected to determine whether the fetus also contains this mutant gene after the pedigree investigation confirms that a certain haplotype in this pedigree is indeed associated with a certain mutant gene. However, sometimes it is impossible to determine certain Haplotype If it is linked to a mutation, then it is impossible to make a correct judgment on its fetus. This is also the disadvantage of RFLP for diagnosis.

DNA of hypervariable region and DNA fingerprint

Human Satellite DNA Or called Suicide DNA It is composed of several short DNA fragments (about 10bp) repeated for many times. Composition and copy number In different individuals and genome Is different in different positions. After extracting genomic DNA from different individuals, four sequences can be identified by their breakpoints Base The restriction endonuclease that does not cut the repeat segment cuts genomic DNA on both sides of the repeat segment, and then carries out gel electrophoresis on the sample. Repetitive fragments of different lengths (mainly due to the different copy numbers of repeat units) will be separated from those containing these repeat sequences Specificity When probes are hybridized, individual specific radioactivity since development Figure, also known as DNA fingerprint 。

Map of DNA fingerprint

Restrictive fragment length polymorphism

It depends on the core sequence of the probe used (i.e. the repeat unit in the repeat sequence). At present, there are two kinds of probes, namely 33.15. Its core sequence is AGAGGTGGGGGCGCGTGGG, and 33.6, namely AGGGCTGGAGG. This means that the two sequences repeat for different times at different positions in the human genome, and the repetition times of the two core sequences at corresponding positions in the genomes of different individuals are also different. In this way, using one of the two probes to hybridize with the human genome DNA fragment cut by suitable enzyme, different DNA fingerprints will be obtained in different individuals, and the DNA fingerprints of probe 33.5 are also different. DNA fingerprint has cell stability and germline stability Mendel Regularly inherited, and Heterozygosity High. Heterozygosity can be understood as follows: point mutation The RFLP caused by a polymorphic There are only two sexual points of view Polymorphism , that is, the tangent point has (+) or the tangent point does not have (-). But because Hypervariable region For RFLP caused by different lengths of repeat fragments genome The number of repeats of the core sequence at a certain location on the top is different in different individuals, such as 10 copies in individual A, 15 copies in individual B, and 18 copies in individual C. Therefore, the number of repeats of the core sequence at the same corresponding position in different individuals is polymorphic, not bimorphic. Even if the number of core sequences at a certain position on the genome is the same Enzyme digestion The length of the core sequence is the same, but the repeat times of the core sequence may be different in other positions of the genome. Because DNA fingerprints are based on Mendel Regularly inherited, the DNA fingerprint of the offspring can be traced back to the DNA fingerprint of their parents, but it is difficult to find the same size on the DNA fingerprint of non parents Suicide DNA Clip. Among the five distinguishable small satellite DNA fragments of the father, four are heterozygous, that is, these four DNA fragments have different individual lengths. Therefore, due to Hypervariable region RLFP caused by different repetition times of core sequence is real Polymorphism 。 In different individuals, this RLFP or DNA fingerprint can be said not to be the same. Even if the DNA fingerprint generated by one probe cannot identify the two individuals, it is possible to distinguish the two individuals by using another probe. DNA Fingerprint technology Has been applied to Paternity test and forensic medicine Confirmation of criminals and other fields.

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