4.2 SSR (Simple Sequence Repeat)
Founded by Moore Equal to 1991. SSR, microsatellite DNA, is a type of DNA tandem repeat consisting of tandem repeats of several (mostly 1 to 5) bases, which are generally short in length and widely distributed in the genome. Different positions, such as (cA)n, (AT)n, (GGC)n and so on. The variability in the number of repeats of different genetic materials leads to a high degree of variability in the length of the SSR, which is the basis for the generation of SSR markers. Although the microsatellite DNA is distributed at different positions of the entire genome, its two-end sequences are mostly conserved single-copy sequences. Therefore, a pair of specific primers can be designed based on the sequences of these two ends, and the core microsatellite DNA sequence between them can be extended by PCR technology. To increase the use of polyacrylamide gel electrophoresis analysis technology to compare the different distances in the plastic sheet to determine the specific SSR locus polymorphism of different organisms, we can obtain the length polymorphism or SSR markers.
The main characteristics of SSR markers are: (1) abundant in quantity, widely distributed in the entire genome; (2) having more allelic variations; (3) co-dominant markers that can identify heterozygotes and homozygotes; (4) ) The experimental repeatability is good and the result is reliable; (5) However, since the sequence information at both ends of the repeated sequence needs to be known when creating a new marker, its development is difficult and the cost is high.
4.3 AFLP (AmpI ified fragment length polymorphism)
AFLP is also known as selective restriction fragmentation amplification (SRFA). The AFLP marker is a polymorphism of the amplified product produced by the selective amplification of genomic DNA fragments. The substance is also the length polymorphism of the restriction enzyme digested fragment, but the polymorphism is The length of the amplified fragment was detected differently. This technology combines the stability of RFLP and the simplicity and high efficiency of PCR technology, while at the same time it can overcome the shortcomings of RFLP banding, small amount of information, and instability of RAPD technology. The basic technical principle and operation steps are as follows: First, genomic DNA is digested with restriction enzymes to form a plurality of random restriction fragments of unequal size; then, specific oligonucleotide connectors are ligated to both ends of these fragments ( (0ligo nuleotideadapter); Then primers are designed based on the linker sequence, due to too many restriction fragments, all the amplification is difficult to separate the product on the gel, for this purpose, add 1 to 3 selective bases at the 3′ end of the primer, and only those The fragment that can pair with the selective base pair can bind to the primer and be amplified as a template to achieve selective amplification of the restriction fragment; finally, these specific amplification products are separated by polyacrylamide gel electrophoresis. Come on.
The main features of AFLP markers are: (1) Because the number of restriction endonucleases and selective bases and numbers that can be used for AFLP analysis are many, the number of markers generated by this technique is infinite; (2) Typical AFLP Analysis, the spectrum of each reaction product between 50 to 100, so a single analysis can detect multiple seats at the same time, and polymorphism is extremely high; (3) co-dominance, a typical Mendelian inheritance; (4) High resolution and reliable results. (5) At present, however, the technology is protected by patents. The kits used for analysis are expensive and require high experimental conditions.
4.4 RAPD (Random amp I polymorphism DNA, randomly amplified polymorphic DNA marker)
RAPD uses PCR technology as the background, and uses artificial oligonucleotides with randomly arranged single-stranded oligonucleotides as primers (9 to 10 bases in length) to perform PCR amplification of the genomic DNA under study, resulting in discontinuities. The DNA product is then electrophoresed to detect DNA sequence polymorphisms. Polymorphisms in these amplified fragments reflect the polymorphism in the corresponding region of the genome.
The RAPD labeling technique uses non-targeted amplification of genomic DNA using random primers (generally 8-10 bases) followed by gel electrophoresis to separate the amplified fragments. If the genomic DNA of the genetic material undergoes DNA fragment insertions, deletions or base mutations in the specific primer binding region, it may cause a corresponding change in the distribution of the primer binding site, resulting in an increase or absence of the PCR product or a change in the molecular weight. RAPD markers are generated if the PCR product is increased or absent.
The main features of RAPD markers are: (1) DNA probes are not needed, and no primers are needed to know the sequence information; (2) Dominant inheritance (very few codominant), heterozygote and homozygote cannot be identified; (3) Technology It is simple and does not involve techniques such as molecular hybridization and autoradiography. The experimental equipment is simple and the cycle is short; (4) The DNA sample needs less (15-25 ng), no radioactivity, the primer is cheap, and the cost is low; (5) but RAPD Marked bands in most markers were dominant, with incomplete information, and poor product stability, poor experimental repeatability, and low reliability.