Recombinant DNA and Its Applications

Recombinant DNA

• Recombinant DNA is a molecule that combines DNA from two sources.
• It is a technology that has been advanced through the Human Genome Project.

Advantages of Recombinant DNA

• One of the primary benefits of recombinant DNA is producing insulin in large quantities for diabetics by placing the human gene for insulin in bacteria.
• The universal property of the genetic code that makes genetically modified organisms possible is the universal nature of the genetic code.

Steps in Recombinant DNA Technology

• The first step in recombinant DNA technology is isolating the gene of interest.
• Restriction endonucleases are used to cut DNA into fragments in recombinant DNA technology.
• Ligation refers to the joining of DNA fragments.
• Cloning the DNA of interest into a vector is done to produce multiple copies of the DNA.
• The final step in the recombinant DNA technology process is recovering and analyzing the cloned DNA.
• Transformation is the term used for the introduction of recombinant DNA into host cells.

Recombinant DNA Technology

• Recombinant DNA technology combines DNA molecules from two sources to create a new molecule.
• The Human Genome Project is associated with the advancement of recombinant DNA technology.
• The primary benefit of placing the human gene for insulin in bacteria is to produce insulin in large quantities for diabetics.
• The universal nature of the genetic code makes genetically modified organisms possible.
• The first step in recombinant DNA technology is isolating the gene of interest.
• Transformation is the term used for the introduction of recombinant DNA into host cells.
• Restriction endonucleases cut DNA into fragments, and ligase enzymes join DNA fragments.

DNA Manipulation

• Gene cloning experiments aim to construct recombinant DNA molecules.
• Cutting and joining DNA are two examples of DNA manipulative techniques.
• Nucleases are enzymes that degrade DNA molecules by breaking phosphodiester bonds.
• Ligase enzymes join nucleic acid molecules together.
• Taq DNA polymerase is commonly used in the polymerase chain reaction (PCR).
• Alkaline phosphatase is an enzyme that removes phosphate groups from the 5' terminus of a DNA molecule.

Restriction Endonucleases and Ligases

• Restriction endonucleases are enzymes that cut DNA at specific sequences.
• Type II restriction endonucleases are most important in gene cloning.
• DNase I is an enzyme that targets any internal phosphodiester bond in a DNA molecule.
• Linkers are short pieces of double-stranded DNA with a known sequence used in DNA manipulation.

PCR and Gel Electrophoresis

• PCR is a technique that amplifies specific DNA sequences.
• Agarose gel is typically used for separating large DNA molecules in electrophoresis.
• Orthogonal field alternation gel electrophoresis (OFAGE) differs from standard gel electrophoresis in that it uses a pulsed field alternating between two pairs of electrodes.
• The migration rate of DNA molecules during electrophoresis is inversely proportional to the size of the molecules.

Protein Purification for Recombinant DNA

• Cell lysis is the first step in the purification of recombinant proteins.
• Affinity chromatography is used to specifically bind and isolate the target protein.
• His-tag, GST-tag, and FLAG-tag are commonly used for purifying recombinant proteins using affinity chromatography.
• Ion-exchange chromatography separates proteins based on their charge.
• SDS-PAGE is used to separate proteins based on their size.

Miscellaneous

• Bacterial DNA undergoes methylation to protect it from restriction endonucleases.
• Restriction endonucleases are involved in degrading foreign DNA in bacteria.
• DNA ligase is used to repair single-stranded breaks in DNA.### Protein Electrophoresis
• Gel electrophoresis is used to separate molecules based on size and charge
• SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) is a type of gel electrophoresis that separates proteins based on their size
• SDS (Sodium Dodecyl Sulfate) denatures proteins and gives them a uniform charge
• The stacking gel in SDS-PAGE concentrates proteins into tight bands
• β-mercaptoethanol helps to break disulfide bonds in proteins
• Coomassie Blue is a common dye used to stain proteins after electrophoresis
• APS (Ammonium Persulfate) catalyzes the polymerization of acrylamide in gel preparation

Gel Filtration Chromatography

• Gel filtration chromatography separates molecules based on size
• It is used to separate proteins of different sizes

Recombinant DNA Technology

• Recombinant DNA technology is used to produce proteins and hormones for biopharmaceuticals
• It involves the use of plasmid DNA as a vector to transfer genes
• Restriction endonucleases are used to cut DNA at specific sequences
• Transformation is the introduction of recombinant DNA into host cells
• Recombinant human growth hormone (rhGH) is used to treat growth hormone deficiency
• Recombinant vaccines are safer and cause fewer side effects than traditional vaccines
• CHO cells (Chinese Hamster Ovary cells) are commonly used in biopharmaceutical production
• Biopharmaceuticals are products derived from living organisms
• Recombinant DNA technology can be used to produce monoclonal antibodies for diagnostic purposes, treating infections, and cancer therapy
• Agrobacterium-mediated transformation is a method used to introduce recombinant DNA into plant cells
• Selectable marker genes are used to identify cells that have taken up recombinant DNA
• Gene therapy aims to replace defective genes with functional ones

Applications of Recombinant DNA Technology

• Producing proteins and hormones for biopharmaceuticals
• Developing recombinant vaccines
• Producing monoclonal antibodies for diagnostic purposes, treating infections, and cancer therapy
• Gene therapy to replace defective genes with functional ones
• Treating growth hormone deficiency with recombinant human growth hormone (rhGH)
• Producing human insulin using recombinant DNA technology
• Treating multiple sclerosis with interferon-beta