Recombinant DNA Overview

Recombinant DNA (rDNA) Definition

• rDNA are DNA molecules formed through laboratory methods of genetic recombination
• This involves combining genetic material from different sources
• rDNA creation bypasses natural genetic combinations
• rDNA is possible because all organisms' DNA shares a similar chemical structure
• Variations lie within the nucleotide sequence

Recombinant DNA (rDNA) Introduction

• Recombinant DNA is a general term for DNA created through combining at least two DNA strands
• Recombinant DNA molecules are also known as chimeric DNA
• This name is for the ability to combine material from different species, similar to mythical chimeras
• rDNA technology uses palindromic sequences to produce sticky and blunt ends of DNA

Recombinant DNA Technology Overview

• Recombinant DNA technology allows isolation and cloning of specific gene or DNA segments
• Creates numerous identical copies
• New genetic combinations are introduced (rDNA) into host cells
• Host cells propagate and multiply the new genetic material, thereby producing identical copies
• The process of this methodology is called Recombinant DNA technology

Obtaining Recombinant DNA (rDNA) - Steps

• Step 1: Isolate the DNA segment to be cloned (the insert)
• Step 2: Cut DNA using specific enzymes (restriction enzymes)
• Step 3: Join the DNA segments together using DNA ligase
• Step 4: Insert the resulting rDNA into a host carrier (a vector)
• Step 5: Allow the DNA to replicate and create identical copies in the host cell
• Step 6: Transfer the modified DNA to an appropriate host cell
• Step 7: Select host cells carrying the rDNA molecule using a marker
• Step 8: Permit rDNA replication to generate a genetically uniform clone collection

rDNA Isolation

• The first step is isolating donor and vector DNA
• Vector DNA isolation method depends on the vector's nature (e.g., bacterial plasmids)
• Plasmids must be purified from the bacterial genome

rDNA Isolation - Techniques

• Ultracentrifugation: Separates plasmid DNA from bacterial genomic DNA based on density differences
• Alkaline Lysis: Denatures genomic DNA at a given alkaline pH, while leaving plasmids intact. Neutralization precipitates the genomic DNA, leaving plasmids in solution.

Cutting DNA - Restriction Enzymes

• Restriction enzymes (e.g., EcoRI) cut DNA at specific sequences creating sticky ends
• This is used to introduce foreign DNA fragments into a vector

Insertion

• Vector: A DNA molecule that replicates inside a host cell and integrates the desired gene of interest.
• Restriction enzymes act as genetic scissors
• Ligase enzyme joins DNA fragments together

Introducing Vector DNA into Host Cells

• Plasmid vectors are added into the host cell culture (e.g., E. coli)
• Calcium ions and heat shock create temporary holes in the host cell membrane
• This allows plasmid DNA to enter

Phage Vectors

• Cloning technique involving bacterial infection utilizing phage vectors
• Phage viruses introduce DNA to a bacterial host
• Growing viruses is more difficult than bacteria or fungi

Placing the Gene in the Vector

• Plasmid DNA are small, making separation by size easy from bacterial chromosomes
• Chromosomal DNA is removed
• The plasmid DNA is then purified

Joining DNA

• Restriction fragments from Donor DNA are mixed with plasmid DNA.
• Sticky ends allow for initial hydrogen bond attraction.
• The sugar-phosphate backbone is joined by DNA ligase.

Recombinant DNA Technology Applications

• Genetic mapping: Mapping genes on DNA.
• Infectious disease/genetic disorder research: Understanding diseases and infections
• Genome sequencing: Determining an organism's complete DNA sequence
• Genetic abnormalities detection: Finding chromosomal disorders, such as Down syndrome
• Disease prevention: Preventing/treating inherited disorders (e.g., sickle cell anemia, phenylketonuria)
• Cellular processes research: Investigating molecular mechanisms underlying biological processes including growth, differentiation, and aging
• Gene therapy: Introducing functional genes to correct defective genes (e.g., sickle cell anemia)
• GMO production: Creating organisms (GMOs or transgenic organisms) with desired traits/features

Negative Aspects of Recombinant DNA Technology

• Erosion of genetic diversity in plants,
• Ecological damage,
• Creation of dangerous toxins/microbes, and
• Potential misuse in biological warfare.

This quiz covers the basics of recombinant DNA (rDNA), including its definition, creation methods, and applications in genetic technology. Explore how rDNA enables the combination of different genetic materials and the significance of this process in modern biology.