What Are Molecular Markers?

The concept of molecular markers is broad and narrow. Broadly defined molecular markers are heritable and detectable DNA sequences or proteins. Narrow molecular markers are specific DNA fragments that reflect certain differences in the genomes of individuals or populations.

Chinese name: molecular marker
Concept: There are broad and narrow senses.

Narrow molecule: Refers to the individual
In the genome: Specific DNA fragment

Molecular marker concept

Molecular markers (Molecular Markers) are genetic markers based on nucleotide sequence variations in genetic material between individuals, and are a direct reflection of DNA-level genetic polymorphisms. Compared with several other genetic markers-morphological markers, biochemical markers, and cytological markers, DNA molecular markers have the following advantages: most molecular markers are codominant, and the selection of recessive traits is very convenient; The genomic variation is extremely rich, and the number of molecular markers is almost unlimited; at different stages of biological development, DNA from different tissues can be used for marker analysis; molecular markers reveal mutations from DNA; appear neutral and do not affect the expression of target traits No linkage with bad traits; the detection method is simple and rapid. With the development of molecular biology technology, there are dozens of DNA molecular marker technologies, which are widely used in genetic breeding, genome mapping, gene mapping, identification of species kinship, gene library construction, gene cloning and so on.

Ideal requirements for molecular markers

The ideal molecular marker must meet the following requirements: have high polymorphism; codominant inheritance, that is, the use of molecular markers can identify heterozygous and homozygous genotypes in diploid; can clearly identify alleles throughout the entire genome; molecular markers are required to be evenly distributed throughout the genome except for special site markers; select neutral (ie, no gene pleiotropy); simple and fast detection methods (such as easy to automate experimental procedures) ; development and use costs are as low as possible; good repeatability in and between laboratories (easy data exchange). However, any molecular marker found is used to separate different biological DNA molecules by electrophoresis, and then hybridized with labeled specific DNA probes to reveal the polymorphism of DNA through autoradiography or non-isotopic coloration technology. .

restriction fragment length polymorphism

(Restriction Fragment Length Polymorphism, RFLP )

In 1974, Grodzicker et al. Established the restriction fragment length polymorphism (RFLP) technology, which is a first-generation genetic marker based on DNA-DNA hybridization. The basic principle of RFLP: the use of specific restriction enzymes to identify and cut the genomic DNA of different organisms, to obtain DNA fragments of different sizes, the number of DNA generated and the length of each fragment reflect the different restriction sites on the DNA Distribution. These fragments are analyzed by gel electrophoresis to form different bands, and then Southern hybridization and radiographic development with cloned DNA probes are performed to obtain RFLP patterns that reflect individual specificity. It represents the difference in length of genomic DNA fragments produced after restriction enzyme digestion. Due to changes in base substitution, rearrangement, and deletion between alleles in different individuals, restriction endonuclease recognition and restriction digestion have changed, resulting in differences in restriction fragment length between genotypes.

RFLP alleles have codominant characteristics. The number of RFLP marker sites is not limited, and the number of gene loci that can be detected is usually 1-4. RFLP technology also has some disadvantages, mainly because it is difficult to clone probes that can express genomic DNA polymorphisms. In addition, the experimental operation is more complicated, the detection cycle is long, and the cost is high. Since the advent of RFLP, it has been widely used in gene mapping and typing, genetic linkage map construction, and genetic diagnosis of diseases.

Variable number of tandem repeat polymorphisms

(Variable Number of Tandem Repeats, VNTR )

A variable number of tandem repeats, also known as minisatellite DNA, is a small repeating DNA sequence, ranging from 10 to several hundred nucleotides, with copy numbers ranging from 10 to 10001. The basic principle of VNTR is roughly the same as RFLP, but it has special requirements for restriction enzymes and DNA probes: Restriction enzyme restriction sites must not be in the repeated sequence to ensure the integrity of the small satellite or microsatellite sequence Sex. The endonuclease has more restriction sites in other parts of the genome, so that the fragment where the satellite sequence is located contains fewer unrelated sequences, and the polymorphism of repeated sequence fragments of different lengths can be fully displayed by electrophoresis. (3) The nucleotide sequence of the DNA probe used for molecular hybridization must be a small satellite sequence or a microsatellite sequence. After molecular hybridization and autoradiography, a large number of small satellite or microsatellite sites can be detected at once, and individual-specific DNA fingerprinting.

The polymorphic information content of small satellite markers is relatively high, ranging from 17 to 19. The disadvantage is the limited number and uneven distribution on the genome, which greatly limits its application in gene mapping. VNTR also has the disadvantages of tedious experimental operation, long detection time and high cost.

Molecular markers

PCR primers for random primers

The nucleotide sequence of the primers used is random, and the amplified DNA region is unknown in advance. The molecular basis for the polymorphism of the DNA segment amplified by random primer PCR is the mutation of the base sequence of the primer binding site on the template DNA amplification segment. Genomes from different sources appear as amplified products on this segment. There were no differences or differences in the size of the amplified fragments. Random primer PCR markers were dominant or codominant.

Randomly amplified polymorphic DNA

(Random Amplified Polymorphism DNA, RAPD )

The RAPD technique was developed in 1990 by William and Welsh et al. Using PCR technology to detect DNA polymorphisms. Basic principle: It uses random primers (generally 8-10bp) to amplify DNA fragments by PCR at non-fixed sites, and then uses gel electrophoresis to analyze the polymorphism of the amplified product DNA fragments. Amplified fragment polymorphisms reflect DNA polymorphisms in corresponding regions of the genome. The primers used by RAPD are different, but for any specific primer, it has its specific binding site on the genomic DNA sequence. Once the genome has inserted, deleted or mutated DNA fragments in these regions, it may cause The distribution of these specific binding sites changes, which results in changes in the number and size of the amplified products and shows polymorphism. For a single primer, it can only detect DNA polymorphisms in specific regions of the genome, but using a series of primers can expand the detection area to the entire genome. Therefore, RAPD can be used to detect polymorphisms in the entire genomic DNA. For constructing genomic fingerprints.

Compared with RFLP, RAPD has the following advantages: simple technology and fast detection speed; RAPD analysis requires only a small amount of DNA samples; does not depend on species specificity and genomic structure, a set of primers can be used for genomic analysis of different organisms; Cost is lower. However, RAPD also has some disadvantages: The RAPD label is a dominant marker that cannot identify heterozygotes and homozygotes; there is a co-migration problem, and gel electrophoresis can only separate DNA fragments of different lengths, not those with the same molecular weight but bases. The sequence consists of different DNA fragments; There are many influencing factors in RAPD technology, so the stability and repeatability of the experiment are poor.

Any primer PCR

(Arbitrarily Primed Polymerase Chain Reaction, AP-PCR )

In the AP-PCR analysis, the primers used are longer (10-50 bp). The PCR reaction is divided into two stages. First, the oligonucleotide primers anneal to the template DNA under low stringency conditions. At this time, some synthesis occurs. To stabilize the template-primer interaction. Then, a cycle of high stringency annealing conditions is performed, and the primer extension of those sequences between the two sites under low stringency annealing conditions can continue to be amplified under high stringency conditions. The PCR product was analyzed by denaturing polyacrylamide gel electrophoresis, and the final reaction result was similar to that of RAPD. As long as the designed primers can reduce artificial production of primers under low stringent annealing conditions, the use of paired combinations of primers can generate new AP-PCR bands, but when the primer pairing is used in combination, the obtained map and single primer can produce The sum of the patterns is very different, so that 50 primers can generate 1,250 different fingerprint patterns.

The AP-PCR method does not require sequence data to be predicted, and the detected genomic samples are arbitrary, and can also be used to detect polymorphisms in near-isogenic lines (or homologous lines). The disadvantage of AP-PCR is that each new polymorphism must be purified for further use. In addition, this method can only distinguish length polymorphisms in heterozygotes.

DNA amplification fingerprinting

(DNA Amplification Fingerprinting, DAF )

DAF is an improved RAPD analysis technology. Unlike RAPD technology, the primers used in DAF analysis are higher in concentration and shorter in length (generally 5 to 8 bp), so it provides more band information than RAPD Much larger, such as when 5 primers are used, the combination of primer and template can amplify about 10 to 100 DNA fragments. PCR amplification products are separated on a gel, and very complex band patterns can be generated by silver staining.

PCR-specific primers

The primers used for PCR labeling of specific primers are designed for DNA segments of known sequences, have specific nucleotide sequences (usually 18-24 bp), and can be amplified at conventional PCR renaturation temperatures for genomic DNA Polymorphism analysis in a specific region.

Sequence marker site

(Sequence Tagged Sites, STS )

STS is a collective name for a class of molecular markers that perform PCR-specific amplification with specific primer sequences. By designing specific primers to bind to specific binding sites in the genomic DNA sequence, they can be used to amplify specific regions in the genome and analyze their polymorphisms. The biggest advantage of using specific PCR technology is that it generates information very reliably, without some ambiguity like RFLP and RAPD.

Simple repeat sequence

(Simple Sequence Repeat, SSR )

Simple repeats (SSR) are also called microsatellite DNAs. The core sequence of tandem repeats is 1 to 6 bp. The most common is double nucleotide repeats, that is, (CA) n and (TG) n. The sequence structure is the same, the number of repeat units is 10 to 60, and its high polymorphism mainly comes from the difference in the number of tandems. The basic principle of SSR markers: design primers based on complementary sequences at both ends of the microsatellite sequence, and amplify the microsatellite fragments by PCR. Due to the different number of tandem repeats in the core sequence, PCR products of different lengths can be amplified by PCR. The amplified products were subjected to gel electrophoresis. The genotype was determined based on the size of the isolated fragments and the allele frequency was calculated.

SSR has the following advantages: (1) A single polyallele is generally detected; microsatellites are codominantly inherited, so heterozygotes and homozygotes can be identified; The amount of DNA required is small. Obviously, when using SSR technology to analyze microsatellite DNA polymorphisms, it is necessary to know the information of the DNA sequences at both ends of the repeated sequence. If you cannot search directly from the DNA database, you must first sequence it.

sequence-specific amplification region

(Sequencecharacterized Amplified Region, SCAR )

The SCAR mark is developed on the basis of RAPD technology. The SCAR marker is to clone the target RAPD fragment and sequence its ends, design specific primers based on the sequences at both ends of the RAPD fragment, and then specifically PCR the gene DNA fragment to identify a single site corresponding to the original RAPD fragment. SCAR markers are dominant, and differences between the DNA to be tested can be directly shown by the presence or absence of amplification products. SCAR labeling is convenient, fast, and reliable. It can quickly detect a large number of individuals with good stability and high reproducibility.

Single primer amplification reaction

(Single Primer Amplification Reaction, SPAR )

SPAR technology is a labeling technology similar to RAPD technology. SPAR also uses only one primer, but the primers used are designed on the basis of SSR. These primers can specifically bind to the spacer sequences between the SSRs, and then the DNA sequences between the SSRs are amplified by PCR technology, and the amplified products are separated by gel electrophoresis to analyze their polymorphisms. In addition, there is another marking technology very similar to SPAR technology, namely ISTR (Inverse Sequence-tagged Repeat) technology. The primers used by ISTR are designed based on inverted repeat sequences. PCR amplification is based on inverted repeat sequences. Between DIVA sequences.

DNA single-strand conformation polymorphism

(Single Strand Conformation Polymorphism, SSCP )

SSCP refers to the conformational variation of single-stranded DNA of the same length due to the difference in the nucleotide sequence, which is manifested as the difference in electrophoretic mobility in non-denatured polypropylene phthalamide. Single-stranded DNA conformational analysis is very sensitive to changes in the DNA sequence, and often a single base difference can be revealed. In SSCP analysis, PCR technology was used to amplify a target fragment in the genomic DNA. The amplified product was denatured. The double-stranded DNA was separated into single strands and separated by non-denaturing polyacrylamide gel electrophoresis. Change the position to determine whether there is a mutation in the target fragment. The SSCP result is judged by comparing multiple samples and observing the position changes between the bands, thereby showing the DNA specificity of different biological individuals, and achieving the purpose of fingerprint analysis. To further improve the detection rate of SSCP, SSCP analysis can be combined with other mutation detection methods. The combination with Heterocluplex analysis (Het) method can greatly improve the detection rate. The Het method uses a probe to hybridize to a single-stranded DNA or RNA to be detected. A hybridization strand containing a pair of base-pair mismatches can be separated from a completely complementary hybridization strand by electrophoresis on a non-denaturing PAG gel. The SSCP and Het analysis of the same target sequence can make the detection rate of point mutation approach 100%, and the experiment is simple.

Double deoxidation fingerprint method

(Dideoxy Fingerprints, ddF )

ddF is an analysis technique combining dideoxy-terminated sequencing and SSCP, which performs SSCP analysis on single-stranded DNA of varying length terminated by dideoxy-terminated. If there is a mutation in the target fragment, all dideoxy-terminated fragments larger than a certain size corresponding to the position of the main mutation have no wild-type system, and there are multiple opportunities for each mutation to detect its mobility change, which improves the efficiency of detecting mutations. The ddF method overcomes the difficulty of affecting the SSCP display due to DNA length during SSCP analysis. A specific single-stranded DNA is produced by a dideoxyglutamic acid, and a DNA fragment of the appropriate length shows SSCP changes.

DNA Molecular marker DNA marker

Amplified fragment length polymorphism

(Amplified Fragment Length Polymorphism, AFLP )

AFLP is a new method for detecting DNA polymorphisms developed by Dutch scientists Zabeau and Vos in 1995. The article was published in Nucleic Acids Research. AFLP is the product of the combination of RFLP and PCR. The basic principle is to first use restriction enzymes to hydrolyze genomic DNA to generate DNA fragments of different sizes, and then to double-strand the artificially-digested fragments of the double-stranded artificial linker as an amplification reaction. The template DNA is then pre-amplified with the complementary strand of the artificial linker as a primer. Finally, 1-3 selective nucleotides are added to the complementary strand of the linker as primers to selectively amplify the template DNA gene. The amplified DNA fragments were separated and detected by acrylamide gel electrophoresis, and polymorphisms were detected according to the length of the amplified fragments. The primer consists of three parts: a core base sequence complementary to the artificial linker, a restriction endonuclease recognition sequence, and a select base sequence (1-10 bp) at the 3 'end of the primer. Several base sequences of the linker and the adjacent restriction fragment are binding sites. The unique feature of this technology is that the special primers used can be used to perform PCR amplification of the digested fragments while knowing the DNA information. In order to make the size distribution of enzyme digestion uniform, two restriction enzymes are generally used. One enzyme has multiple digestion points and the other has a small number of digestion points. Therefore, AFLP analysis mainly produces fragments digested by two enzymes. . AFLP combines the advantages of both RFLP and RAPD technologies, with the advantages of high resolution, good stability, and high efficiency. However, its technology is expensive and has high requirements on DNA purity and endonuclease quality. Although AFLP technology was born short, it can be called another major breakthrough in molecular labeling technology, which is considered to be a very ideal and effective molecular labeling.

Digestion amplified polymorphic sequence

(Cleaved Amplified Polymorphism Sequences, CAPS )

CAPS technology is also called PCR-RFLP, which is essentially a method combining PCR technology with RFLP technology. The basic principle of CAPS is to design a set of specific PCR primers (19-27bp) using the DNA sequence resources of known sites, and then use these primers to amplify a DNA fragment at the site, and then use a specific The resulting amplified product is cleaved by a restriction enzyme, and the fragments are separated by gel electrophoresis, stained and analyzed by RFLP. CAPS markers reveal information on restriction length variations of specific PCR fragments. CAPS is a class of co-dominant molecular markers, which has the advantage of avoiding the film transfer step in RFLP analysis, while maintaining the accuracy of RFLP analysis. In addition, since many restriction enzymes can be digested with amplified DNA, there is a greater chance of detecting a polymorphism.

DNA Molecularly labeled DNA chip

Single Nucleotide Polymorphism (SNP)

SNP marker is the third generation DNA genetic marker proposed by American scholar Lander E in 1996. SNP refers to the difference in individual nucleotides or only small insertions or deletions between different alleles at the same site. Detecting differences in single nucleotides at the molecular level, SNP markers can help distinguish genetic material differences between two individuals. The human genome appears approximately every 1250 bp SNP, and more than 2,000 markers have been mapped to the human chromosome, which is of great significance for human genomics research. The best method for detecting SNPs is DNA chip technology. SNP is called the third generation DNA molecular marker technology. With the development of DNA chip technology, it is expected to become the most important and effective molecular marker technology.

Molecular marker genetic marker

A special easy-to-recognize expression of a genotype. Its two most basic characteristics are:

Heritability.

Recognizability (polymorphism).

Genetic markers are an indispensable tool for studying genetic phenomena and species evolution.

Application fields of molecular markers

Genomic Mapping and Gene Mapping

For a long time, the genetic maps of various organisms have been constructed based on conventional markers such as morphology, physiology, and biochemistry. The genetic maps are limited to a small number of organisms, and the resolution of the maps is mostly low and the map distance is large. Low saturation and limited application value. The use of molecular markers for genetic map construction is one of the major advances in the field of genetics. With the development of new marker technology, new members on the list of biological genetic maps will continue to increase, and the density of marks on maps will also become higher and higher. By building a complete high-density molecular map, you can locate genes of interest.

Cloning genes based on maps

Map cloning (Map-bascd cloning) is a new gene cloning technology developed in recent years with the successive establishment of molecular marker genetic maps and gene molecular mapping. Using molecular marker-assisted map-based cloning, you can directly clone genes without knowing the sequence of the gene or understanding the expression product of the gene. Map-based cloning is the most common method for gene identification, at least in theory, it applies to all genes. High-density genetic maps, large-scale physical maps, large-segment genomic libraries, and complete genome sequences provided by genomic research have paved the way for the widespread application of map-based cloning.

Application of species kinship and systematic classification

Molecular markers are widely present in various regions of the genome. By comparing the polymorphisms of molecular markers randomly distributed throughout the genome, it is possible to comprehensively assess the diversity of research subjects and reveal their genetic nature. Using genetic diversity results, species can be clustered to understand their phylogeny and kinship. The development of molecular markers provides a powerful means for studying species kinship and systematic classification.

For disease diagnosis and genetic disease linkage analysis

In 1980, Bostein and others successfully applied PFLP technology to the diagnosis and analysis of sickle anemia, pioneering genetic diagnosis. PFLP is inherited in the Mendelian way, so it can be used as a genetic marker of disease-causing gene loci on the chromosome. Many linked disease-causing genes were mapped. Small satellites and microsatellites are widely used for diagnosis of diseases and linkage analysis of genetic diseases because of their high polymorphism. With the advent of high-throughput SNP detection technology and methods, as the largest number of polymorphic markers that are easy to detect in batches, SNPs are used in linkage analysis and gene mapping, including genetic mapping of complex diseases, association analysis, and individual and population environmental pathogenic factors And susceptibility to drugs will play an increasingly important role.

Prospects of molecular marker technology

Molecular marker technology has developed rapidly and is widely used in genetic research of animals and plants. Molecular markers have been used in many breeding and production of plants and animals, such as corn, soybeans, chickens, and pigs, and they have focused on applied research in gene mapping, assisted breeding, and disease treatment. Some application results have been achieved. The development of molecular marker technology is a hot topic in the field of molecular biology. With the rapid development of molecular biology theory and technology, molecular marker technology with faster analysis speed, lower cost and greater information content will be developed. The combination of molecular marker technology with extraction extraction programming, electrophoretic film analysis automation, and computerization of information (data) processing will definitely accelerate the construction of genetic maps, gene mapping, gene cloning, identification of species relationships, and human-related disease-causing genes. Diagnosis and analysis.

The picture shows the silver staining test results of AFLP