Gene cloning technology.

. Objective The
of
generefers to the gene to be studied or applied, that is, the gene to be cloned or expressed. Obtaining the gene of destination is the most important step in the process of molecular cloning.
There are several methods currently used to obtain the gene of the purpose, such as restricted endoenzyme direct separation method, library screening method, insotroplification method and synthetic method, among which the restrictive endoenzyme method directly isolates the gene and polyenzyme chain reaction (
). PCR
) or reverse transcription-polyenzyme chain reaction (
RT-PCR
) in-body amplification purpose
DNA
fragments are currently the most commonly used method.
(i) the direct separation of the gene in the destination using restrictive enzymatic
. 1. The preparation of the primary nucleogen from the primary nuclear genome is relatively small, with several restrictive endoenzymes to digest the primary nuclear genome, or with some kind of restrictive endoenzyme to partially digest the genome to be studied, you can obtain a variety of fragments of varying sizes, some of which will contain the gene of purpose, these fragments will be inserted into the vector for cloning, screening, you can obtain the desired gene of destination.
2. The preparation of the ceutron genome from the ceutron genome is relatively large, and the number of clones that need to be screened after digestion directly with restrictive endoenzymes is too large to operate. A restrictive endoenzyme can be used to digest genomic DNA, produce continuous DNA fragments, and then electrophoresis to isolate these DNA fragments, and then a specific probe to interbreed with these DNA fragments, in areas containing specific templates will appear hybrid belt, can be initially identified whether the genome contains the gene of purpose.
We can also clone all the enzymatically cut genomic DNA fragments into appropriate vectors, make genome libraries, and then hybridize them with specific probes with different clones in the genome library, which means that cloned DNA fragments contain specific gene sequences. Sequencing positive clones, separating the next clone fragment at the end of the first clone fragment, and then using overlapping sequences between DNA fragments to identify other clones.
step by step and eventually get the entire gene sequence, a technique called chromosome walk. (ii) Using PCR or RT-PCR methods to prepare the target genedesign and synthesize the quotation according to published gene sequences, using PCR (using genomic DNA as a template) or RT-PCR (using mRNA as a template) to obtain the target gene fragments from
tissues
or cells for gene operation, which is the most commonly used method in the laboratory to obtain known genes.
One of the great advantages of using PCR to obtain the destination gene is that the destination gene can be modified with appropriate enzyme cut points, starting cocoons or terminating cocoons, etc. on the lead sequence as needed, or by altering the base sequence (artificial
mutation
) through human mismatch.
For unknown genes, unknown genes can be obtained by building an RT-PCR library: using mRNA end poly A sequence tail design common quotations, all mRNA reversals into
cDNA
, using various tool enzymes to tail the end of cDNA, and then using a pair of common citations to amplification of all cDNA fragments, pcR amplification fragments into the appropriate vector, the construction of PCR-cDNA library.
advantage of this method is that it can amplification of mRNA with low expression levels, which can help to screen out genes with low expression abundance. Because of PCR amplification, the accuracy of the gene sequence needs to be further determined by other methods.
There is also a way to obtain the gene of interest by using computer technology for "computer cloning", that is, using genetic information in GenBank, using software to compare similarities (esogens) between different genus genes, using conservative area sequences to design quotations, and then using PCR or RT-PCR to obtain unknown gene fragments from different genus or tissue cells. This is a shortcut to a new gene that is currently possible.(iii) Construction and screening of genomic libraries or cDNA libraries insert genomic DNA into appropriate vectors with restrictive endoenzymes to obtain clone vectors containing different insertion fragments, a mixture of which contains genome fragments of different lengths, which is the gene library.
if all mRNAs in cells are reversed into cDNA and then all cDNA fragments are cloned into appropriate vectors to form a clone vector mixture containing different cDNA fragments, this is the cDNA library. Many tissue or cell genomes or cDNA libraries are now available from commercial companies.
When a genome library or cDNA library is obtained, specific probes can be synthesized from known information, and gene fragments of interest are screened from the library using
nucleic acid
molecular hybridization, which is still a common means of obtaining new genes. The construction and use of genomic libraries or cDNA libraries can be found in the appropriate sections of this book.
.