Primer design and synthetic experiment.

primer design and synthetic experiment.

1, [123 primer design and synthetic experiment. polymerase chain reaction, PCR technology It is a method of rapidly amplifying a particular gene or DNA sequence in vitro, so that the in vitro amplification method is also known. PCR technology has become one of the use and extensive means of molecular biology research, and primer design is a crucial ring in PCR technology. It is easy to cause experimental failure using an inappropriate PCR primer: expressed as an amplified project A plurality of strips outside (such as forming the primer dimer), no band or weak, and so on. Most of the PCR primer design can be carried out through computer software, and the template sequence can be submitted directly to a particular web page, which is designed to be designed, and the primer design professional software can be operated on the local computer. The primer design principles are as follows: 1, the primer should be designed and specific in the conservative region of the sequence. The primer sequence should be located at the height of the genomic DNA, and there is no ordered sequence of non-amplified regions. This reduces the non-specific binding of the primer and the genome to improve the specificity of the reaction;

2, the length of the primer is generally 15-30 bp. Commonly used is 18-27 bp, but should not be greater than 38, because the excess length causes its extended temperature than 74 ° C, not suitable for TAQ DNA polymerase reaction;

3, the primer should not form a secondary structure. The primer dimer and the hairpin structure are too high (more than 4.5 kcal / mol) to cause primer dimer tape, and reduce the effective concentration of the primer and the PCR reaction cannot be carried out normally; [123

4, the GC content of the primer sequence is generally 40-60%. Evergreen or too low is not conducive to reactions. The GC content of the upper and lower proders cannot be different;

5, the TM value of the corresponding template position sequence in the primer can make the replication conditions in the corresponding template position sequence. . There are a variety of ways for the TM value, such as according to the formula Tm u003d 4 (G + C) +2 (A + T);

6, primer 5 u0026 # 39; Terminal sequence affects the PCRToo big, therefore is often used to introduce the modified site or marker. The corresponding sequence of the carrier of the PCR product can be inserted according to the next experiment.

7, primer 3 u0026 # 39; end is not modified. The primer 3 u0026 # 39; the end base of the terminal has a great influence on the DNA synthesis efficiency of TAQ enzymes. Different end bases lead to different amplification efficiency in the mismatch position, the mismatch efficiency of the last base A is significantly higher than the other 3 bases, and therefore should be avoided 3 u0026 # 39 of the primer; A.

8, the primer sequence itself or the primer cannot be increased in more than 3 consecutive bases, such as GGG or CCC, increase the errors.

9, a g value refers to the free energy required for DNA double strand formation, which reflects the relative stability of the base pair in the double-stranded structure. . 3 u0026 # 39 should be selected; the terminal G value is low (no more than 9), while 5 u0026 # 39; end and intermediate G value relatively high primers. The 3 u0026 # 39 of the primer is too high, which is easy to form a double-strand structure in the mismatch site and trigger DNA polymerization;

is worth mentioning Yes, the difficulty of primer design of various templates is different. Some templates themselves are difficult, such as high GC content or low, resulting in very suitable primers that are not found to find various indicators; PCRs used as clonal derivements, because the product sequence is relatively fixed, and the selection of primer design is more free. Low, in this case, you can only return it, try to meet the conditions. Service features:

1. Provide reference sequences, design principles, and analysis reports for primer design, so that you have a understanding of your primer or probe's performance and characteristics for your understanding and analysis of experimental results.

2. Supply and post-processing before amplification, including, including nucleic acid extraction, PCR, nucleic acid purification, Sequence analysis, data statistics, etc.

3. A service, The guarantee of the whole process.