Recombinant DNA Technology Overview

Recombinant DNA Technology (Background)

• Recombinant DNA is the general name for combining a piece of one DNA strand with another DNA strand
• It's also sometimes called a "chimera"
• The most common method combines DNA from two different organisms
• There are three different methods for making recombinant DNA: Transformation, Phage Introduction, and Non-Bacterial Transformation

Transformation

• Select a piece of DNA to be inserted into a vector

• Cut the selected DNA with a restriction enzyme

• Ligate the DNA insert into the vector with DNA ligase

• The inserted DNA contains a selectable marker for identifying recombinant molecules

• An antibiotic marker is often used; host cells without a vector die when exposed to the antibiotic; host cells with the vector live because they are resistant

• The vector is inserted into a host cell, which is transformation

• E. coli is an example of a host cell; host cells must be specially prepared

Non-Bacterial Transformation

• In microinjection, DNA is directly injected into the nucleus of the cell being transformed
• In biolistic, host cells are bombarded with high-velocity micro-projectiles (gold or tungsten particles coated with DNA)

Historical Background

• Restriction enzymes were discovered in 1968 by Werner Arber.
• Type II restriction enzymes were discovered in 1969 by Hamilton O. Smith.
• Daniel Nathans advanced the technique of DNA recombination, showing type II enzymes could be useful in genetic studies (1970-71)
• Paul Berg developed methods for splitting DNA molecules and attaching other DNA segments (1973)
• Stanley N. Cohen and Herbert W. Boyer first inserted recombined genes into bacterial cells in 1973

DNA Methylation

• Methylation adds a methyl group to a DNA base
• This can affect gene expression and health

Steps in Recombinant DNA Technology

Isolation of Genetic Material

• The first step is isolating the desired DNA from other macromolecules
• Since DNA exists within the cell membrane with other macromolecules (RNA, polysaccharides, proteins, and lipids), it must be separated and purified
• This often involves enzymes like lysozyme, cellulose, chitin, ribonuclease, and proteases
• Ethanol is used to precipitate the DNA

Restriction Enzyme Digestion

• Restriction enzymes act as molecular ‘scissors’ cutting DNA at specific locations
• They incubate the purified DNA with a selected restriction enzyme at optimal conditions
• The technique 'Agarose Gel Electrophoresis' is used to separate DNA fragments based on size after the digestion

Amplification Using PCR

• Polymerase Chain Reaction (PCR) is a method for making multiple copies of a DNA sequence
• It amplifies a single copy or a few copies of DNA into thousands or millions
• Key components of PCR include template (DNA to be amplified), primers (small oligonucleotides complementary to a region of the DNA), enzyme (DNA polymerase), and nucleotides

Ligation of DNA Molecule

• The purified DNA and the vector of interest are cut using the same restriction enzyme
• The ends are joined together using DNA ligase, forming a hybrid DNA molecule.
• Hybrid DNA molecule is also called a recombinant DNA molecule
• The technology behind this process is called recombinant DNA technology

Insertion of Recombinant DNA into Host

• Recombinant DNA is introduced into a recipient host cell (often a bacterial cell) This process is called transformation
• Bacterial cells are treated to become competent to take up foreign DNA, such as using thermal shock, Ca++ ion treatment, electroporation, or other methods.

Isolation of Recombinant Cells

• The transformation process creates a mixed population of transformed and non-transformed host cells
• Selection techniques filter out only the transformed host cells
• Marker genes (e.g., in plasmid vectors like PBR322, including Ampicillin and Tetracycline resistant genes) are used to distinguish recombinant cells from non-recombinant cells

Application of Recombinant DNA Technology (example)

• Scientific research
• Medicine and pharmacology industry
• Pest or insecticide-resistant plants
• Bacteria used to clean oil or toxic spills
• Food industry