PCR (Polymerase Chain Reaction)

Often called a "revolution in the study of DNA", the PCR, or Polymerase Chain Reaction was created by Kary B. Mullis. He was awarded the Nobel Prize for Chemistry for invention of the PCR.

The PCR is like "molecular photocopying" - using it, scientists and researchers can amplify - or, copy - DNA, quickly and efficiently.

 In PCR, a sample is first heated in order to denature DNA: this is basically separation of the DNA molecule into two pieces of single-stranded DNA. Then, an enzyme called "Taq polymerase" is used to synthesize, or build, two new strands of DNA using the original two strands as templates. Thus, the original DNA is duplicated, and each of the new molecules now contains an old and a new strand of DNA. Then, each of these synthesized strands can be used to create two new copies, and then more copies from those copies, and so on. The whole process is repeated from 30 to 40 times.

The process yields billions of exact copies of the same DNA molecule. The whole process takes only a few hours and is automated; a machine called a thermocycler can be programmed to adequately denature and synthesize the DNA molecules according to the quantities of DNA being made. Using PCR, scientists can replicate DNA quickly in order to test developed gene therapies or even to study the effect of a gene therapy on a DNA molecule.(Citation 31)(Citation 32)(Citation 33)

Vectors 

Vectors are entities used to transfer genes from one organism to another. Gene therapy involves treatment at the DNA or chromosome level - vectors are usually around the size of DNA itself or at least specially designed. There are two main types of vectors: viral vectors, and non-viral vectors. Vectors include retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, liposomes, and naked DNA.

Viral vectors have a variety of advantages.

• Some types of viral vectors can be engineered to target specific types of cells in the body.
• Viral vectors can be genetically modified  to prevent them from replicating and destroying the target cell.
• Viruses are made from nature; they are a small representation of desired ideal bio-particles.

At the same time however, viruses have a few disadvantages.

• Viruses can trigger immune reactions in patients (due to their viral/disease causing nature). Some patients, conversely, may be immune to infection by a type of virus and thus, the treatment may not work.
• There is sometimes also a risk for viral infection if the viral DNA has not been properly removed from the virus.
• Viruses, being non-living, cannot expand to carry larger genes.

Non-viral vectors include circular DNA molecules, called plasmids, and other natural or artificially developed vectors. Unlike viral vectors, most non-viral vectors do not cause immune responses.

Plasmids, used as non-viral vectors, work the same way as they do naturally in bacteria. Bacteria exchange plasmids (as circular DNA molecules) with other bacteria through methods such as conjugation. Scientists use these plasmids to insert a desired gene, such as a healthy gene meant to replace a faulty gene. into the plasmid DNA. The plasmid is then utilized.(Citation 34)

Another prominent nonviral vector is a nanoparticle. Nanoparticles are developed using nanotechnology, the science of things smaller than 100 nanometers in scale. Nanoparticles are artificially created polymers. Advances in nanotechnology as nonviral gene delivery mechanisms have yielded significantly positive results. For example, as shown in the visual on the bottom right side, genetic analysis assays can be completed using specialized nanoparticle delivery methods.

Also shown below to the left is a possible gene drug delivery method through the use of biosynthetic nanoparticles.

 A possible/theoretical nano-particle designed to deliver a gene therapy. Note that the particle is designed in such a way as to be most organic as possible; the presence of a lipid bilayer pseudo-genetic material make it a striving parallel to the actual cells it will be delivered to.

Video Citation2

 Viruses can be used in a variety of ways to deliver specific gene therapies to patients.

 A type of viral vector: adenovirus. Using adenoviruses, desired DNA can be quickly moved into the cell. The virus is already designed by nature itself for efficient entry into the cell.

 Using plasmids to transfer DNA from one organism to another. Like viruses, plasmids are best at what they do in Nature: transferring genetic material.

 How can nanoparticles be used to aid in gene therapy? Well, nanoparticles have an extremely small size. Such small sizes, combined with great potential for customization, make nanoparticles a cutting-edge solution to the use of vectors in gene therapy