Molecular biology cloning

Molecular cloning is one method in molecular biology that is commonly used to amplify a genetic sequence of interest. This is accomplished by inserting recombinant DNA into a vector which can then carry DNA fragments in host organisms to be amplified. This process of amplification is based on molecular biology standard, first is to recombine the target gene into the vector DNA molecules in vitro. Then transfer the recombinant DNA to host cells. After transferring, there is a screening of cells which have expressed the recombinant DNA, after purification and amplification.

Molecular Biology Cloning Technology Process:

    • Isolate the target gene and vector:
      1. Direct separation is suitable for the extraction and separation of bacterial chromosomes, plasmids and virus DNA whose genetic background are of interest to be studied.
      2. Gene synthesis is used to generate short DNA fragments whose sequence is known clearly.
      3. cDNA can be synthesized by reverse transcription from mRNA.
      4. Screening the gene of from the genomic library for molecular cloning.
    • The target gene and vector are cleaved with a restriction enzyme.

This allows the fragments to be more easily connected later.

    • The target gene and vector are then ligated with DNA ligase.

This seals the connection between target gene and vector.

    • Transfer the ligated recombinant vector into host cells

Bacteria: E. coli, fungi: Yeast, insect cells or mammalian cells.

    • Conduct screening at different levels using different methods to test for quality.

For example: vector size, enzyme digestion results, screening markers and so on.

Molecular biology cloning generally uses DNA sequences from two different organisms. First is the species that is the source of the DNA to be cloned. Second is the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning technology is central to many contemporary areas of modern biology and medicine.

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