- CHARITH AKLANSS DODANGODAGE -
What is DNA cloning?
❑ By definition, the general term “cloning” means obtaining multiples copies of something, let it be individuals of an animal species, or molecules. DNA cloning is a Molecular Biology technique that allows us to get multiple copies of identical sequence-specific double-stranded DNA fragments, for various purposes.
❑ This process takes of the fact that a single cell can be induced to take up and replicate a single recombinant DNA molecule.
❑ The concept of DNA cloning was born around 1970.
❑ How to Clone - DNA exists as a double helix of antiparallel polymer strands with the nucleotide units in each joined by 50 –30 phosphodiester bonds. The ability to selectively break and join these bonds is the basis of DNA cloning.
❑ Large amounts of DNA are needed for genetic engineering. Multiple copies of a piece of DNA can be made either by using polymerase chain reaction (PCR) or by cloning DNA in cells. ❑ Virtually any DNA sequence can be cloned and amplified, but there are some factors that might limit the success of the process. Such as;
✓ DNA sequences that are difficult to clone are inverted repeats
✓ Origins of replication
✓ Centromeres and telomeres
✓ There is also a lower chance of success when inserting large-sized DNA sequences
✓ Inserts larger than 10kbp have very limited success
How is DNA cloning done?
Here are the things you need to do to clone DNA.
❑ A source of the DNA of interest: normally, a bulk amount of the DNA of interest is necessary in the form of tissue, bacterial culture, etc. From this, the DNA is extracted through conventional extraction techniques.
❑ Plasmids: In order to be able to get multiple DNA copies, we need a self-replicating DNA molecule capable to carry the DNA fragment of interest. These DNA molecules are called plasmids, and they exist naturally in several microorganisms. These plasmids can be obtained in multiple copies per cell, being therefore possible to get substantial amounts of DNA to work with.
❑ Restriction enzymes: DNA cloning is possible because it can be cut at specific places (sequence-specific) thanks to a large set of proteins called “restriction enzymes” naturally appearing in many bacteria.
❑ Cells: We need living cells in which to introduce plasmids and amplify its amount. Normally bacteria are used, very convenient also thanks to its growth rate. Here are the things you need to do to clone DNA.
Basic steps of DNA cloning:
❑ Extraction of DNA thanks to DNA & RNA Extraction Kits.
❑ Restriction analysis and use of restriction enzymes to cut the suitable fragment of DNA to be cloned
❑ Cutting the plasmid with the suitable restriction enzymes in order to be able to insert in it the desired fragment.
❑ Use of enzymatic DNA modifying enzymes (e.g. T4 DNA Ligase) to get a whole, circular plasmid-DNA insert (functional plasmid) capable of self-replication.
❑ Introduction of the plasmid into the cell by means of a technique called transformation. ❑ Culture of cells harboring the plasmid to amplify its amount.
❑ Selection of cells carrying the plasmid and the insert in culture plates by means of antibiotics and chromogenic substances, indicating the presence of a plasmid and a DNA fragment. ❑ Restriction or sequence analysis to check the cloned DNA sequence
❑ DNA cloning takes advantage of the fact that the chemical structure of DNA is fundamentally the same in all living organisms.
❑ Therefore, if any segment of DNA from any organism is inserted into a DNA segment containing the molecular sequences required for DNA replication, and the resulting recombinant DNA is introduced into the organism from which the replication sequences were obtained, then the foreign DNA will be replicated along with the host cell's DNA in the transgenic organism.
❑ DNA cloning is similar to polymerase chain reaction (PCR) in that it permits the replication of DNA sequence. The fundamental difference between the two methods is that molecular cloning involves replication of the DNA in a living microorganism, while PCR replicates DNA in an in vitro solution, free of living cells.
❑ Whatever combination of host and vector are used, the vector almost always contains four DNA segments that are critically important to its function and experimental utility:
✓ DNA replication origin is necessary for the vector (and its linked recombinant sequences) to replicate inside the host organism
✓ one or more unique restriction endonuclease recognition sites to serves as sites where foreign DNA may be introduced
✓ a selectable genetic marker gene that can be used to enable the survival of cells that have taken up vector sequences
✓ a tag gene that can be used to screen for cells containing the foreign DNA
❑ DNA cloning is used to create a large number of copies of a gene or other piece of DNA. The cloned DNA can be used to:
✓ Work out the function of the gene
✓ Investigate a gene’s characteristics (size, expression, tissue distribution)
✓ Look at how mutations may affect a gene’s function
✓ Make large concentrations of the protein coded for by the gene
Where can DNA cloning be used?
❑ Protein production: If the cloned DNA codifies for a protein, large amounts of bacteria are grown in industrial fermenters, from which the desired protein can be purified. These proteins are normally biopharmaceuticals, like for example, insulin.
❑ Genetic analysis: Having a greater amount of DNA makes possible to analyze genetic material in detail, to study DNA transcription, translation of transcripts, DNA profiling in search of specific sequences ,amplification of very small amounts of genetic material, etc.
❑ Gene therapy: Specific DNA sequences can be used to produce proteins directly into cells in which defective genes are present causing a genetic disease. This set of techniques is referred to as “gene therapy” and allows treating previously untreatable diseases.
❑ Genotyping: This is the process of determining the DNA sequence specific to an individual's genotype. This process can be accomplished by several techniques, such as high-resolution melt (HRM) analysis, or any other mutation detection technique. All these techniques will provide an insight into the individual's genotype, which can help determine specific sequences that can be manipulated and cloned for further analysis.
Introduction of PCR
# What is PCR?
❑ Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies of a particular region of DNA.
❑ Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro.
❑ PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.
❑ In PCR, the reaction is repeatedly cycled through a series of temperature changes, which allow many copies of the target region to be produced.
❑ Typically, the goal of PCR is to make enough of the target DNA region that it can be analyzed or used in some other way. For instance, DNA amplified by PCR may be sent for sequencing, visualized by gel electrophoresis, or cloned into a plasmid for further experiments.
❑ PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.
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