Genetic Engineering: Restriction Endonucleases and DNA Manipulation

Chapter 10: Genetic Engineering and Genetic Analysis

Introduction

This section introduces the fundamental tools and concepts used in genetic engineering, focusing on the role of restriction endonucleases in manipulating DNA. These enzymes are essential for cutting DNA at specific sites, enabling scientists to splice, dice, and reverse nucleic acids for various applications in microbiology and biotechnology.

Restriction Endonucleases

Definition and Function

• Restriction endonuclease: An enzyme that recognizes specific sequences of DNA and cleaves the phosphodiester bonds between adjacent nucleotides within or near those sequences.

• These enzymes are often referred to as "molecular scissors" because they can cut DNA at precise locations.

• They are commonly used to cleave DNA at desired sites, which is crucial for genetic engineering techniques such as cloning, gene insertion, and recombinant DNA technology.

Recognition Sequences and Palindromes

• Restriction endonucleases recognize palindromic sequences in DNA. A palindromic sequence is a segment of double-stranded DNA in which the nucleotide sequence reads the same in both directions (5' to 3' and 3' to 5').

• For example, the sequence 5'-GAATTC-3' is palindromic because its complementary strand is 3'-CTTAAG-5', which reads the same in the opposite direction.

• Cleavage at these sites produces DNA fragments with either "blunt" or "sticky" (overhanging) ends, which are useful for further manipulation.

Examples of Restriction Endonucleases

Several well-known restriction enzymes are used in molecular biology. The following table summarizes three common enzymes, their recognition sequences, and their cutting patterns:

Endonuclease	Recognition Sequence	Cutting Pattern
EcoRI	5'-GAATTC-3' 3'-CTTAAG-5'	Cuts between G and A: 5'-G↓AATTC-3' 3'-CTTAA↑G-5'
HindIII	5'-AAGCTT-3' 3'-TTCGAA-5'	Cuts between A and A: 5'-A↓AGCTT-3' 3'-TTCGA↑A-5'
HaeIII	5'-GGCC-3' 3'-CCGG-5'	Cuts between G and C: 5'-GG↓CC-3' 3'-CC↑GG-5'

Mechanism of Action

• Restriction enzymes scan the DNA molecule and bind to their specific recognition sequence.

• Once bound, the enzyme cleaves the DNA backbone at or near the recognition site, resulting in DNA fragments.

• The type of ends produced (blunt or sticky) depends on the enzyme and its cutting pattern.

Applications in Microbiology and Genetic Engineering

• Restriction endonucleases are essential for recombinant DNA technology, allowing scientists to insert, remove, or replace genes in various organisms.

• They are used in cloning, gene mapping, and genetic analysis.

• Sticky ends produced by some enzymes facilitate the ligation of DNA fragments from different sources, enabling the creation of genetically modified organisms (GMOs).

Example: DNA Cleavage by HindIII

• When the restriction enzyme HindIII acts on its recognition sequence, it cuts the DNA to produce fragments with overhanging "sticky" ends, which can be joined with complementary sequences from other DNA molecules.

Additional info: Restriction enzymes are naturally found in bacteria, where they serve as a defense mechanism against invading viral DNA by cutting it into nonfunctional pieces.