DNA recombination is referred to as the making of a functional composite DNA strand that is obtained from two or three fragments from more than one organism. The composite DNA is often called recombinant DNA. This is made possible through genetic engineering. An aspect of genetic engineering known as molecular cloning makes recombinant DNA production possible.
- 0.1 Molecular Cloning
- 0.2 DNA Extraction
- 0.3 DNA Digest / DNA Cutting
- 0.4 DNA Splicing
- 0.5 Induced Transformation
- 0.6 Screening and selection of transformed bacteria
- 0.7 Recombinant Proteins
- 1 Description of DNA Recombination Quiz
In molecular cloning procedure, the following are to be considered:
- Choice of host organism and cloning vector;
- Preparation of vector DNA;
- Preparation of DNA to be cloned;
- Creation of recombinant DNA;
- Introduction of recombinant DNA into the host organism;
- Selection of organisms containing recombinant DNA;
- Screening for clones with desired DNA inserts and biological properties.
The host organisms are usually bacteria, Escherichia coli is the most preferred bacteria host in molecular cloning due to its shorter replication time. The cloning vectors are mostly plasmids (pBR322, pU8, etc).
Molecular cloning process starts with DNA extraction which involve selecting of broth medium of interested microorganism or cell (in case of eukaryotes) and treating them with lysozyme to degrade the cells followed by the addition of protease to degrade the protein components of the intracellular components of the cells, addition of RNAse to degrade the RNA components and purification by centrifugation. The figure below represents the schematic process of DNA extraction from cells
DNA Digest / DNA Cutting
This is done by treating both the extracted DNA and cloning vector with the same restriction enzyme. There are a series of restriction enzymes, but the most used is EcoRI. The restriction enzyme cut both the cloning vector (plasmid) and the extracted DNA at a particular sequence giving rise to sticky ends which are easily joined together by DNA ligase towards the end of the experiment.
The digested DNA fragment is polymerized using the appropriate primer, under appropriate temperature condition.
The mixture of the polymerized DNA fragment, the cloning vector, and DNA ligase are mixed. Fragments having the same sticky ends as the cloning vector are annealed by DNA ligase.
Escherichia coli preferably is used as the host for carrying the cloning vector. A mixture of the bacterial culture and cloning vector are mixed and then and then subjected to heat shock in the presence of divalent calcium chloride, or by using electroporation, cell squeezing or gene gun technique.
Screening and selection of transformed bacteria
Most plasmids do have antibiotic resistance gene marker. Bacteria are plated on culture media containing the genes coded for in the cloning vectors, survivors are said to have transformed and are selected and sub-cultured. The final screening stage is the culturing of the survivor bacteria on a culture media on which the gene-spliced into the cloning vector can be expressed in the form of recombinant protein. For example, if a bacteria carrying a cloning vector with a gene for insulin is plated on a medium containing Isopropyl β- d-1-thiogalactopyranoside (IPTG) which allows the expression of insulin.
The product of the successful transformation of bacteria is known as a recombinant protein. Molecular cloning technology has been utilized in the commercial production of pharmaceutical products such as recombinant chymosin, recombinant clotting factor vii, recombinant hepatitis vaccine, recombinant human growth hormone, and recombinant insulin among others. Genetic Modified Foods (GMO) such as golden rice that is rich in beta- carotene and vitamin A, as well as disease resistant plants, has also been discovered through the knowledge of DNA recombination.
- INTRODUCTION TO MOLECULAR BIOLOGY
- MECHANISM OF DNA REPLICATION
- TRANSCRIPTION IN CELL BIOLOGY
- TRANSLATION IN CELL BIOLOGY
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Description of DNA Recombination Quiz
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DNA recombination is referred to as the making of a functional composite DNA strand that is obtained from two or three fragments from more than one organismCorrect
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Molecular cloning process starts with DNA extractionCorrect
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Most plasmids have antibiotic resistance gene markerCorrect
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