Screening and identification of gene sequence cloning.

. NetworkSection VI Screening and identification of gene sequence cloningDestination Sequence and Vector
DNA
the efficiency of proper connection, recombination of imported cells are not 100 percent, so the final growth and reproduction of cells and different with the purpose sequence. Typically, a vector carries only a certain piece of external DNA, and a cell accepts only one recombined DNA molecule. Finally
only
, if any, of the cell populations that are cultured are recombinants (recombinants) that contain the sequence of purpose. Screening the target recombinant is equivalent to obtaining a clone of the destination sequence, so screening (dcreening) is an important step in gene cloning. In the construction of vectors, the selection of host cells, the design of molecular cloning scheme must carefully consider the problem of screening. The basic principles of common techniques are described below. , according to the recombinant carrier signs for screening the most common carrier carries signs of resistance, such as anti-ammonia penicillin (anpr), anti-teteycline (terr), anti-kanr and so on. When
medium
contains
antibiotics
, only cells carrying the corresponding drug-resistant gene vector can survive and reproduce, which sieves out all cells that fail to receive vector DNA. If the exonymic sequence is inserted into the vector's drug-resistant gene to inferfy the drug-resistant gene, the resistance marker disappears. For example, the prolific pBR322 contains two drug-resistant genes, anpr, and terr, if the purpose sequence is inserted into the terr gene sequence, the conversion of E. coli, so that bacteria in amino penicillin or terecin-containing mediums, where no cells receiving the granule DNA can grow; Bacteria that can grow in both ammonia penicillin and tycycline contain proton pBR322, but pBR322 is not inserted into the destination sequence, and bacteria that can grow in ammonia penicillin and cannot grow in tycycline are likely to have recombination primers containing the destination sequence.contains lacZ's blue-white screening method, which has been widely used in recent years. For example, the purpose sequence is inserted into the aforementioned polyclonal point of the primer pUC19, converted into E. coli, cultured in a medium containing ammonia penicillin, IPTG, X-gal, where it can grow and appear white colonies, its bacteria are likely to contain recombinative primer inserted into the destination sequence, so that it is easy to obtain the clone of the destination sequence.According to the logo of the recombinant vector to screen, you can screen out a large number of non-purpose recombinants, but only rough sieve, for example, bacteria may mutate and cause changes in resistance, but does not represent the insertion of the purpose sequence, so further detailed screening is required., nucleic acid hybridization methodusing the labeled nucleic acid as a probe and the DNA of the converted cells for molecular hybridization, can be directly screened and identified for sequence cloning. The commonly used method is to copy the converted growth of the bacteria to the nitric acid fiber membrane, with alkali-cracking bacteria, the DNA released by the bacteria is adsorbed on the membrane, and then with the labeled nucleic acid probe temperature-breeding hybridization, nucleic acid probe will be combined with the destination sequence of the bacteria DNA without being washed away. Nucleic acid probes can be marked with radionuclides, combined with radionucleic acid probes of the fall group can be indicated by radiological self-development (auroradiography) method, nucleic acid probes can also be marked with non-radioactive material, usually by color to indicate the location, so that the bacteria containing the sequence of destination can be selected., the
pcR
methodthe emergence of PCR technology to clone screening has added a new means. If the length of the desired sequence and the sequence at both ends are known, a pair of quotations can be designed to be amplified by converting the DNA from the cell as a template, and if the expected length of the PCR product is obtained, the converted cell may contain the sequence of the purpose.,
immunology
using specific
antibody
and the specific binding of the target gene expression product for screening. This method does not directly screen the gene of the purpose, but indicates the transformational cell containing the gene of the purpose by reacting with the gene expression product, thus requiring the experimental design to enable the gene of the purpose to be able to express its encoded product when it enters the subject cell. Antibodies can be used by specific enzymes such as peroxidase, alkaline
phosphatase
and so on, binding to the enzyme label antibody, the enzyme can catalyz a specific substrate decomposition and render color, thus indicating the location of the cell set containing the target gene. Immunological methods are highly specific and sensitive, and are suitable for screening cell clones with few genes of purpose from a large number of transformed cell collections., DNA restrictive endoenzyme map analysis this is further analysis after the above screening. The purpose sequence insertion vector causes changes in the vector DNA restrictive enzyme map, e.g. a 600bp long destination sequence is inserted into the multiclonal point of pUC19 using the sticky end connections at both ends of it, the recombinant protons are increased to 3.3kb, with Eoor I and Sal I double enzymatic cutting will appear 600bp and -2.7kb two pieces of DNA, extract the metastatromat DNA of the converted bacteria for enzymatic cutting and then do electrophoresis to observe its enzyme cutting map, you can analyze the results, such as inserted in the purpose sequence there are other restrictive endoenzyme bits, can also be observed on the enzyme cutting electrophoresis map. This allows further identification of the recombination body is not the intended purpose of cloning., nucleotide sequence determinationobtained by the purpose sequence or gene cloning, should be determined with its nucleic acid sequence to final identification. The nucleic acid cloning of a known sequence is confirmed by sequence determination, and the nucleic acid clone of an unknown sequence needs to be determined in order to determine its structure and infer its function, and further research is used. Therefore, nucleic acid sequence determination is an essential identification step in molecular cloning. The principles and methods of nucleic acid sequence determination are described in detail in the experimental teaching materials.
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