Recombinant DNA Technology Overview

Recombinant DNA Technology

• Recombinant DNA (rDNA) is artificial DNA created by combining two or more DNA sequences that would not normally occur together through gene splicing.
• Recombinant DNA technology enables the artificial production of DNA. This technology has been used to alter DNA in living organisms and may have even greater practical applications in the future.
• The technology was developed by Boyer and Cohen in 1973.
• Stanley N. Cohen received the Nobel Prize in Medicine in 1986 for his work on growth factors.
• Herbert Boyer helped construct the first recombinant DNA using bacterial DNA and plasmids.
• Recombinant DNA technology is a branch of medical science currently under research in an organized and dedicated effort.
• Recombinant DNA is combined from two different DNA sources into a single molecule. This procedure is often useful when the artificial DNA is reproduced, known as DNA cloning.
• The process involves isolating DNA, cutting DNA segments using restriction endonucleases, joining the fragments using DNA ligase, inserting the rDNA into a host cell, replicating and expressing DNA, and then identifying the host cells containing the rDNA.
• Cloning vectors are plasmids, bacteriophages, viruses, or artificial chromosomes. Vectors are often used for replicating DNA within a host cell.
• Most functional vectors have multiple cloning sites (MCS) or poly linkers.
• There are different ways of getting a target gene including genomic DNA, artificial synthesis, PCR amplification, or RT-PCR.
• A polymerase chain reaction (PCR) technique can amplify DNA from small samples.
• Reverse transcription polymerase chain reaction (RT-PCR) is a variant of PCR and uses reverse transcriptase to first make DNA complements (cDNA) from RNA, then amplifies the cDNA using traditional PCR methods.
• Cloning vectors are vital for this process as they allow DNA to be replicated autonomously within a compatible host cell.
• Cloning vectors often have genetic markers or genes for selecting host cells that have successfully taken up foreign DNA. The cells that include the desired recombinant genes are often screened to identify them.
• Recombinant proteins produced in prokaryotic or eukaryotic cells can be used for therapeutic purposes. Recombinant proteins can be used in various medical and biological applications.
• Some challenges involve mRNA processing in mammals that does not occur in bacteria; this can be addressed by chemically synthesizing genes containing exons or making cDNA using processed mRNA.
• Bacteria may not perform post-translational modifications, so only proteins that do not undergo this process are synthesizable in bacteria.

Gene Cloning (Molecular Cloning)

• The process of producing numerous copies of a DNA segment.
• Essential for studying genes and producing large quantities of specific DNA sequences.

Types of Vectors

• Plasmid vectors
• Bacteriophage vectors
• Virus vectors

Types of Blotting Techniques

• Southern blotting: For DNA molecules
• Northern blotting: For RNA molecules
• Western blotting: For proteins

Transgenic Organisms

• Organisms that contain genes from other organisms
• Genetically modified organisms (GMOs) can produce crops with genetically desirable traits, making them resistant to pesticides/herbicides, enhancing production, adding vitamins to improve nutritional content, or increasing shelf life.
• Transgenic animals also include those that express genes from other organisms for various applications, like producing therapeutic proteins.
• Animals may be modified for research or for producing proteins with therapeutic value.

Application in Medicine

• Human gene therapy
• Diagnosis of genetic disorders
• Forensic evidence

Ethical Concerns with Recombinant DNA Technology

• The use of recombinant DNA technology raises concerns about potential dangers including the risks of introducing allergens or causing harm to wild populations from genes that spread through pollination. Research and development costs can also be significant, creating complications for large-scale implementation when considering human use and profit.

Other Applications of Recombinant DNA technology

• Producing hormones for other therapeutic uses.
• Producing safer vaccines.