How is the chemical synthesis of gene separation done?

The chemical synthesis of DNA fragments is a very mature and simple technology. According to the predetermined nucleotide sequence of the DNA fragment to be synthesized, four kinds of nucleotide monomers can be automatically connected into oligonucleotide fragments through 3 ′→ 5 ′ phosphate bonds by using a DNA synthesizer. At present, this method is often used to synthesize primers, oligonucleotide linkers and gene fragments.

According to the nucleotide sequence determined by the gene or deduced from the amino acid sequence of protein, the corresponding gene fragments can be chemically synthesized. The difference is that the DNA fragments that make up genes are generally very long. Firstly, several DNA fragments of about 200bp must be chemically synthesized according to the nucleotide sequence of the gene, and then the DNA fragments containing the complete target gene are reassembled by enzymatic connection.

It mainly includes two methods to synthesize gene fragments: phosphodiester method and phosphotriester method. The basic principle of phosphodiester method is to connect two deoxymononucleotides with proper protective bases at the 5'- or 3'- ends to form a deoxydinucleotide with phosphodiester bond; The principle of phosphite triester method is to connect the 3'- end of the oligonucleotide chain to be synthesized with insoluble carriers such as porous glass beads (CPG) through 3'-OH, and then add nucleotide monomers from 3'-5' direction in turn. The active functional groups of the used nucleotide monomers are all protected, and a cyclic synthesis reaction is divided into four steps: deprotection, coupling reaction, capping reaction and oxidation.

The ability to chemically synthesize oligonucleotide fragments is generally limited to 150200bp, but the size of most genes exceeds this range, so it is necessary to synthesize shorter oligonucleotide fragments from several longer genes and then assemble them into a complete gene, which often makes the preparation of genes more difficult, and the cost of chemically synthesizing genes is relatively high, so this method is mainly suitable for preparing target genes with known nucleotide sequence and small molecular weight. For the synthesis of longer genes, chemical synthesis of oligonucleotides can be combined with enzymatic synthesis of DNA according to the gene or amino acid sequence, or artificial synthesis of genes can be carried out. The specific operation is as follows: firstly, a plurality of oligonucleotides containing 80 100 nucleotides are chemically synthesized; There is an overlapping sequence of 1924 nucleotides between each oligonucleotide fragment; Then the oligonucleotides were mixed in the same amount (molar concentration), and under the action of DNA polymerase (or stress: role = italictaqemphasis enzyme), the single-stranded part was complementary to the double-stranded part by sequence: overlap: extension (SOE-PCR). DNA degenerates into single strand, and there is still an overlapping sequence of 1924 nucleotides between each single strand oligonucleotide fragment. These two single-stranded parts were complemented by SOE-PCR to obtain double-stranded.