Screening and identification of target gene clones-Gene Therapy

The efficiency of correctly connecting the target sequence with the vector DNA and the efficiency of recombinant introduction into cells are not 100%, so the cells that grow and reproduce in the end do not all carry the target sequence. Generally, a carrier only carries a certain segment of exogenous DNA, and a cell only accepts one recombinant DNA molecule. Only a portion, or even a very small portion, of the cultured cell population is a recombinant containing the target sequence. Screening out the target recombinant is equivalent to obtaining a clone of the target sequence, so dcreening is an important step in gene cloning. When constructing vectors, selecting host cells, and designing molecular cloning schemes, careful consideration must be given to screening issues.

The basic principles of commonly used techniques are introduced below.

1、Screening based on the marker of the recombinant vector

　 The most common markers carried by carriers are resistance markers, such as resistance to ampicillin (anpr), resistance to tetracycline (terr), resistance to kanamycin (kanr), etc. When antibiotics are present in the culture medium, only cells carrying the corresponding resistance gene vector can survive and reproduce, which eliminates all cells that cannot receive the vector DNA. If the exogenous target sequence is inserted into the resistance gene of the vector to inactivate the resistance gene, the resistance marker will disappear.

For example, plasmid pBR322 contains two drug resistance genes, anpr and terr. If the target sequence is inserted into the terr gene sequence and transformed into E. coli, the bacteria will be placed in a medium containing ampicillin or tetracycline. Cells that have not received plasmid DNA will not be able to grow; Any bacteria that can grow in both ampicillin and tetracycline contain plasmid pBR322, but its pBR322 has not been inserted into the target sequence. Any bacteria that can grow in ampicillin but cannot grow in tetracycline are likely to be recombinant plasmids containing the target sequence.

The blue white screening method containing lacZ 'in the carrier has been widely used in recent years. For example, inserting the target sequence into the multi cloning site of plasmid pUC19 mentioned earlier, transforming E. coli, and culturing it in a medium containing ampicillin, IPTG, and X-gal. Any colony that can grow and appear white is likely to contain recombinant plasmids inserted with the target sequence, making it easy to obtain clones of the target sequence.

Screening based on the markers of the recombinant vector can remove a large number of non target recombinants, but it is still a rough screening. For example, bacteria may mutate and cause changes in drug resistance, but it does not represent the insertion of the target sequence, so further detailed screening is needed.

2、Nucleic acid hybridization method

Using labeled nucleic acids as probes for molecular hybridization with transformed cell DNA can directly screen and identify target sequence clones. The commonly used method is to copy the transformed bacterial colonies onto a nitrocellulose membrane, use alkaline bacteria to adsorb the DNA released by the colonies onto the membrane, and then incubate and hybridize with labeled nucleic acid probes. The nucleic acid probes bind to the bacterial colony DNA containing the target sequence without being eluted. Nucleic acid probes can be labeled with radioactive isotopes, and colony groups combined with radioactive nucleic acid probes can be indicated by radiourography. Nucleic acid probes can also be labeled with non radioactive substances, usually by color indicating the location, so that colonies containing the target sequence can be selected.

3、 PCR method

The emergence of PCR technology has added a new means to clone screening. If the length of the target sequence and the sequences at both ends are known, a pair of primers can be designed and synthesized to amplify the DNA obtained from transformed cells. If the expected length of PCR product can be obtained, the transformed cells may contain the target sequence.

4、 Immunological methods

Screening is performed by utilizing the specific binding of specific antibodies to target gene expression products. This method does not directly screen the target gene, but indicates the transformed cells containing the target gene through the reaction with the gene expression product. Therefore, it requires experimental design to enable the target gene to express its encoded product after entering the receptor cell. Antibodies can be labeled with specific enzymes such as peroxidase and alkaline phosphatase. When combined with enzyme labeled antibodies, the enzyme can catalyze the decomposition of specific substrates and present a color, indicating the location of cell colonies containing the target gene. Immunological methods have strong specificity and high sensitivity, and are suitable for screening a few cell clones containing the target gene from a large number of transformed cell aggregates.

5、 DNA restriction enzyme mapping analysis

The insertion of the target sequence into the vector will cause changes in the restriction map of the vector DNA. For example, if a 600bp target sequence is inserted into the multi clone site of pUC19 using the sticky end connections cut by EooR I and SalI at both ends, the recombinant plasmid will increase to 3.3kb. After double enzyme digestion with Eoor I and SalI, two DNA fragments of 600bp and -2.7kb will appear. The plasmid DNA of the transformed bacteria can be extracted, digested, and analyzed by electrophoresis to observe its restriction map; If there are other restriction enzyme sites in the inserted target sequence, they can also be observed on the enzyme digestion electrophoresis map. This can further identify whether the recombinant is the intended clone.

6、 Nucleotide sequence determination

The cloned target sequence or gene obtained must be identified using its nucleic acid sequence determination for final identification. Nucleic acid clones with known sequences need to undergo sequence determination to confirm the accuracy and correctness of the obtained clones; Nucleic acid clones with unknown sequences need to be sequenced in order to determine their structure and speculate their function, which requires further research. Therefore, nucleic acid sequencing is an essential identification step in molecular cloning.