Recombinant DNA Technology Overview

Recombinant DNA Technology

• Recombinant DNA technology, also known as gene cloning, genetic engineering, or molecular cloning, is a collection of protocols.
• The goal of these manipulations is to introduce a gene from one organism into a host cell, where it replicates and is studied.
• DNA is extracted from a source organism and treated enzymatically.
• Fragments of DNA are joined to a cloning vector to form a recombinant DNA molecule (construct).
• DNA constructs are transferred into a host cell.
• Typically, a single DNA construct enters each host cell, though efficiency of uptake is low.
• Host cells with the desired DNA construct need to be identified and separated from those without it.
• The desired insert DNA of a construct can be characterized after isolation.
• In some cases, the insert DNA is expressed in the host cell, and the encoded protein can be harvested.

Restriction Endonucleases

• These are DNA cutting enzymes found in bacteria.
• Restriction enzymes limit the types of bacteriophages able to infect bacteria.
• Each restriction enzyme recognizes a short, specific sequence of nucleotide bases.
• These recognition sequences are called recognition sequences and are randomly spaced throughout the DNA.
• Bacteria protect their own DNA from degradation by modifying their recognition sequences using methylases; these add methyl groups to adenine or cytosine bases within the recognition sequence.
• Recognition sequences are often palindromic, meaning the sequence reads the same on both strands in 5´to 3´ direction.
• Type II restriction enzymes are commonly used in laboratories as they cut DNA within their recognition sequence.

Plasmids and Vectors

• Plasmids are small, circular, double-stranded DNA molecules found in bacteria.
• Plasmids exist independently from chromosomal DNA and replicate autonomously.
• Plasmids provide bacteria with genetic advantages, such as antibiotic resistance.
• Plasmids can range in size from a few thousand to hundreds of thousands of base pairs in length.
• Synthetic plasmids are also called vectors in molecular biology.
• Vectors are engineered plasmids, which serve as vehicles enabling replication of desired DNA sequences.
• Vectors contain several characteristics that make them useful for molecular biologists including an origin of replication, a multiple cloning site, and selectable markers to enable identification of cells carrying the desired gene.

Shuttle Vectors

• Some plasmids can exist in multiple species.
• These plasmids are called shuttle vectors.
• Shuttle vectors can be used to move cloned DNA sequences among various organisms.
• Shuttle vectors are useful for genetically modifying multicellular organisms, such as inserting herbicide resistance genes in plants.