It's possible to clone whole organisms such as Dolly the sheep, but DNA cloning is different. It uses molecular biology techniques to make identical copies of DNA sequences or single genes.
Using genetic engineering methods, segments of the DNA genetic code are identified and isolated. DNA cloning then copies the nucleic acid sequences in the segments.
The resulting identical copies can be used for further research or for biotechnology applications. Often the gene that is copied encodes a protein that can form part of medical treatments. DNA technology including DNA cloning is supporting the understanding of how genes work and how the genetic code of humans influences the functioning of the body.
DNA Cloning: Definition and Process Overview
DNA cloning is the molecular biology process of making identical copies of DNA segments located in the chromosomes that contain the genetic code of advanced organisms.
The process generates large quantities of the target DNA sequences. The aim of DNA cloning is to produce the target DNA sequences themselves or to produce the proteins encoded in the target sequences.
The two methods used in DNA cloning are called plasmid vector and polymerase chain reaction (PCR). In the plasmid vector method, DNA strands are cut using restriction enzymes to produce DNA fragments, and the resulting segments are inserted in cloning vectors called plasmids for further duplication. The plasmids are placed in bacterial cells that then produce the DNA copies or encoded proteins.
In the PCR method, the segment of DNA strands to be duplicated is marked with enzymes called primers. A polymerase enzyme makes copies of the marked part of the DNA strand. This method doesn't use restriction enzymes and can produce cloned DNA from small samples. Sometimes the two DNA technology methods are used together to incorporate the best features of each in an overall reaction.
The Plasmid Vector Method
The vector of the method refers to the plasmid used to hold the target DNA segment to be cloned. Plasmids are small circular strands of non-chromosomal DNA found in many organisms including bacteria and viruses.
Bacterial plasmids are the vector used for inserting the target DNA segment into bacterial cells for further duplication.
Selecting and isolating the target DNA: Before the DNA cloning process can begin, the DNA sequences have to be identified, especially the beginnings and the ends of the DNA segments.
Such DNA sequences can be found by using existing cloned DNA with known sequences or by studying the protein produced by the target DNA sequence. Once the sequence is known, the corresponding restriction enzymes can be used.
Cutting the target DNA with restriction enzymes: Restriction enzymes are selected to look for the DNA code at the beginning and end of the target sequences.
When the restriction enzymes find a special coded sequence of base pairs called restriction sites, they attach themselves to the DNA at that location and wind themselves around the DNA molecule, severing the strand. The cut DNA segments containing the target sequence are now available for duplication.
Choosing the plasmid vector and inserting the target DNA: A suitable plasmid ideally contains the same DNA coding sequences as the DNA strand from which the target DNA was cut. The circular DNA strand of the plasmid is cut with the same restriction enzymes as were used for cutting the target DNA.
A DNA ligase enzyme is used to promote DNA segment linking, and the ends of the target DNA segment link up with the cut ends of the plasmid DNA. The target DNA now forms part of the circular plasmid DNA strand.
Inserting the plasmid into a bacterial cell: Once the plasmid contains the DNA sequence to be cloned, the actual cloning can take place using a process called bacterial transformation. The plasmids are inserted into a bacterial cell such as E. coli, and the cells with the new DNA segments will start producing copies and the corresponding proteins.
In bacterial transformation, the host cells and plasmids are incubated together at body temperature for about 12 hours. The cells absorb some of the plasmids and treat them as their own plasmid DNA.
Harvesting the cloned DNA and proteins: Most plasmids used for DNA cloning have antibiotic resistance genes incorporated in their DNA. As the bacterial cells absorb the new plasmids, they become resistant to antibiotics.
When the culture is treated with antibiotics, only those cells that have absorbed the new plasmids survive. The result is a pure culture of bacterial cells with cloned DNA. That DNA can then be harvested or the corresponding protein can be produced.
The PCR (Polymerase Chain Reaction) Method
The PCR method is simpler and copies existing DNA in place. It doesn't require cutting with restriction enzymes or inserting plasmid DNA sequences. This makes it especially suitable for cloning DNA samples with a limited number of DNA strands. While the method can clone DNA, it can't be used for production of the corresponding protein.
Uncoiling the DNA strands: DNA in chromosomes is tightly coiled in a double helix structure. Heating the DNA to 96 degrees Celsius in a process called denaturation makes the DNA molecule uncoil and separate into two strands. This separation is required because only a single strand of DNA can be cloned at one time.
Selecting the primers: As with plasmid vector DNA cloning, the DNA sequences to be cloned have to be identified with special emphasis on the beginnings and ends of the DNA segments. Primers are enzymes that attach to specific DNA code sequences, and they have to be selected to mark the target DNA segments. The right primers will attach to the DNA molecule sequences to mark the beginnings and ends of the target segments.
Annealing the reaction to bind the primers: Cooling the reaction down to about 55 degrees Celsius is called annealing. As the reaction cools, the primers are activated and attach themselves to the DNA strand at each end of a target DNA segment. The primers act only as markers, and the DNA strand does not have to be cut.
Producing identical copies of the target DNA segment: In a process called extension, the heat-sensitive TAQ polymerase enzyme is added to the reaction. The reaction is then heated to 72 degrees Celsius, activating the enzyme. The active DNA polymerase enzyme binds to the primers and copies the DNA sequence between them. The initial DNA sequencing and cloning process is complete.
Increasing the yield of cloned DNA: The initial annealing and extension process creates relatively few copies of the available DNA strand segments. To increase the yield through additional DNA replication, the reaction is cooled again to re-activate the primers and let them bind to other DNA strands.
Then, re-heating the reaction activates the polymerase enzyme again and more copies are produced. This cycle can be repeated 25 to 30 times.
Using the Plasmid Vector and PCR DNA Cloning Methods Together
The plasmid vector method relies on an ample initial supply of DNA to cut and insert into plasmids. Too little original DNA results in fewer plasmids and a slow start to cloned DNA production.
The PCR method can produce a large quantity of DNA from few original DNA strands, but because the DNA is not implanted in a bacterial cell, protein production is not possible.
To produce the protein encoded in the DNA fragments to be cloned from a small initial DNA sample, the two methods can be used together, and they can complement each other. First the PCR method is used to clone DNA from a small sample and produce many copies.
Then the PCR products are used with the plasmid vector method to implant the produced DNA into bacterial cells that will produce the desired protein.
Examples of DNA Cloning for Biotechnology
Molecular biology uses gene cloning and DNA replication for medical and commercial purposes. The bacteria with cloned DNA sequences are used to produce medicines and replace substances that people with genetic disorders can't produce themselves.
Typical uses include:
- The gene for human insulin is cloned in bacteria that then produce the insulin used by diabetics.
- Tissue plasminogen activator is produced from cloned DNA and used to help prevent blood clots.
- Human growth hormone can be produced and administered to people who can't produce it themselves.
Biotechnology also uses gene cloning in agriculture to create new characteristics in plants and animals or enhance existing characteristics. As more genes are cloned, the number of possible uses increases exponentially.
Examples of DNA Cloning for Research
DNA molecules make up a small fraction of the material in a living cell, and it is difficult to isolate the influences of the many genes. The DNA cloning methods deliver large amounts of a specific DNA sequence for studying, and the DNA is producing proteins just as it did in the original cell. DNA cloning makes it possible to study this operation for different genes in isolation.
Typical research and DNA technology applications include examining:
- Function of a gene.
- Mutations of a gene.
- Gene expression.
- Gene products.
- Genetic defects.
When more DNA sequences are cloned, it is easier to find and clone additional sequences. The existing cloned DNA segments can be used to determine whether a new segment matches the old one and which parts are different. Identifying a target DNA sequence is then faster and more accurate.
Examples of DNA Cloning for Gene Therapy
In gene therapy, a cloned gene is presented to the cells of an organism whose natural gene is damaged. A vital gene that produces a protein required for a specific organism function could be mutated, changed by radiation or affected by viruses.
When the gene doesn't work properly, an important substance is missing from the cell. Gene therapy tries to replace the gene with a cloned version that will produce the required substance.
Gene therapy is still experimental, and few patients have been cured using the technique. The problems lie with identifying the single gene responsible for a medical condition and delivering many copies of the gene to the right cells. As DNA cloning has become more widespread, gene therapy has been applied in several specific situations.
Recent successful applications have included:
- Parkinson's disease: Using a virus as a vector, a Parkinson's disease-related gene was injected into the midbrains of patients. Patients experienced improved motor skills without any adverse side effects.
- Adenosine deaminase (ADA) deficiency: A genetic immune disorder was treated by removing patients' blood stem cells and inserting the ADA gene. Patients were able to produce at least some of their own ADA as a result.
- Hemophilia: People with hemophilia don't produce specific proteins that help blood clot. A gene for the production of one of the missing proteins was inserted in the liver cells of patients. Patients produced the protein and bleeding incidents were reduced.
Gene therapy is one of the most promising applications of DNA cloning, but other new uses are likely to proliferate as more DNA sequences are studied and their function is determined. DNA cloning delivers the raw material for genetic engineering in the quantities needed.
When the role of genes is known and their proper function can be assured through replacement of defective genes, many chronic diseases and even cancer can be attacked and treated at a genetic level using DNA technology.
- NCBI Bookshelf: Molecular Cell Biology (4th Edition): DNA Cloning With Plasmid Vectors
- Boston University: Introduction: Cloning (DNA Ligation) & Transformation
- University of Nebraska: Gene Cloning
- North Dakota State University: Polymerase Chain Reaction (or PCR)
- University of Utah: Genetic Science Learning Center: Gene Therapy Successes