Since the human genetics laboratory Mullis of PE-Cetus Corporation in the United States invented the epoch-making polymerase chain reaction (PCP0) in 1985, PCR has become one of the most basic and important technical means in molecular biology-[I. However, whether a pair of suitable nucleotide fragments can be used as primers to effectively amplify the template DNA sequence will undoubtedly determine the success or failure of PCR. Now animal genetics and breeding has entered the molecular era, seeking to affect animal genetic tables at the genetic level The new type of gene highlights the importance, so primer design has undoubtedly become the top priority in finding new genes.
1 Primer design and preliminary screening
Primer design and preliminary screening are basically completed through some molecular biology software and related websites. Currently, the software Primer Premier 5 or a free online PCR primer design program provided by the Whitehead Institute of Biomedicine Research Center on the Internet on the Internet Primer 3 to design primers, and then use the software Oligo 6 for primer evaluation, you can initially get a set of relatively satisfactory primers. But for beginners, using software and programs to design primers seems to be impossible to start. In fact, as long as we master the basic principles and precautions of primer design, all problems will be solved. Because whether it is software or program, these basic principles and precautions are used as default standards for primer design. Therefore, when we design primers, we don't need to spend too much time thinking about the parameters of software and programs. If there are no special requirements, we can set some parameters as default values. Below we mainly discuss the principles and precautions of primer design.
â‘ The length of the primer is generally 15-30 bp, preferably 18-24 bp, because too short and easy to form mismatch (False priming) to reduce specificity, and too long will also reduce specificity and reduce yield [21.
â‘¡Primers are preferably single within the template, which means that there is no mismatch within the template. Especially at the 3 'end, it is necessary to avoid more than 4 consecutive base complementary mismatches.
â‘¢The GC content of the primer sequence is preferably between 40% and 60%, and the difference in the GC content of the upstream and downstream primer sequences should not be too large. The last 5 bases at the 3 'end should not be rich in GC, especially 3 consecutive G Or C.
â‘£ The free energy AG required for DNA double-strand formation should decrease from the 5 'end to the 3' end. The AG at the 3 'end should preferably not be higher than 9.0 keaf mol [31.
⑤ Avoid the formation of stable primer dimers (Dimer and Cross DimeO and hairpin structure), when AG is higher than 4.5 keal / mol, it is easy to cause the above two structures.
â‘¥ The template region where the primer is located should be located in the exon region, preferably spanning an intron region, so as to facilitate the functional identification and phenotypic analysis of the amplified fragments.
⑦ If primers are designed using DNA as a template, the product length is ideal in 100-600 bp. When using mRNA as a template to design primers, the product length is ideally 150-300 bp.
⑧ The 5 'end has little effect on PCR, and modification sites and markers can be introduced [2].
As long as we have mastered the above principles and precautions, we can select a few pairs of target primers we need from a set of primers designed by software and programming. Primer Premier 5 and Oligo
2 Secondary screening of primers
The secondary screening of primers refers to further selecting the pair of primers suitable for our specific and high-efficiency PCR amplification from the first few pairs of primers selected. At this step, the following two points should be noted. First, the series of primers obtained are tested in Genebank. That is, each primer is searched for homology in the blastnr of the comparison tool, and the primers with higher homology to other parts of the genome are discarded, that is, primers that may form a mismatch. In general, homology of more than 10 bp in succession may form a relatively stable mismatch, especially the 3 'end of the primer should avoid consecutive homology of 5-6 bp. Second, when designing primers using mRNA as a template, first use the knowledge of bioinformatics to roughly judge the splice sites of exons and introns (such as the GENESCAN tool at http://CCR-081.mit.edu/GENESCAN.html Or GeneParser is soft, and then discard the primer that is exactly at the splice site.
3 Final evaluation of primers
The primers obtained after our initial screening and secondary screening can be used for synthesis. After synthesis, we can perform final evaluation of the primers through PCR amplification. One is the specificity and efficiency of PCR amplification. Is it possible to obtain specific bands after optimization of PCR conditions, that is, no extra bands other than the target bands. In addition, whether the amount of PCR products is sufficient, that is, the phenomenon that bands and bands are very weak. Second, when using DNA as a template to design primers, whether the PCR amplification product is the same size as the expected PCR product. If the difference is too large, G is more than 100 b, it may be a mismatch product. The third is whether to form primer dimer band.
We can identify the success or failure of primer design by combining the final primer evaluation and sequencing results, and accumulate valuable experience for our future primer design.
4 Primer design considerations when isolating new genes using comparative genomics
The primers obtained after the first screening and the second screening when amplifying known genes can basically meet the requirements, but when using comparative genomics to isolate new genes, the following two points should be noted when designing primers: â‘ the choice of template. If primers are designed using DNA as a template, first find the gene in other species that is homologous to the new gene to be isolated in Genebank. Use the Blast tool and Clustalw tool to compare the homology of the retrieved genes. According to the principle of comparative genome positioning, select the DNA sequences of conserved functional genes with deep research and densely labeled human and mammals (such as mice) to design primers [51, The primer segment must be absolutely conserved among species, the difference should not be greater than 2 bp, especially the 3 'end must be completely homologous. If the EST contig of the new gene of the species to be isolated obtained by computer cloning strategy is used as a template to design primers, the homology between the ESTs and the information probe must be greater than 80%, the length should be greater than 100 bp, and avoid the EST junction Design primers at possible splicing sites for exons and introns. â‘¡The primer sequence is best located in the adjacent exon region and at least 25 bD away from the splice junction between the exon and the intron, so as to facilitate the functional identification and phenotypic analysis of the amplified fragment.
[references]
[1] H. A. Edited by Edhi, translated by Tian Ding. The principle and application of PCR technology-DNA amplification [c]. Beijing: United Press of Peking Union Medical College, Beijing Medical University, 1991.
[2] Edited by Lin Wanming. PCR Technology Operation and Application Guide [c]. Beijing: People's Military Medical Press, 1993.
[3] Oligo software help file.
[4] Rychlik, W. and Rhoads, R. E. A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplifi-cation of DNA [J]. Nucleic Acids Res, 1989, 17: 8543-8551.
[5] Yu Mei. Isolation, identification and physical location of four new genes on pig chromosome 12. Huazhong Agricultural University, 2002
Rooftop Turf,Artificial Turf On Roof,Green Fake Grass Rooftop,Artificial Roof Grass
Changshu Keyuan Eco-friendly New Materials Co.,Ltd , https://www1.ailiqingky.com