IGEM:IMPERIAL/2007/Tutorials/Guide for Engineers/Standard Lab Techniques

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Contents

Introduction

The Molecular Biologist Toolbox Kit

Restriction Enzymes

Restriction enzymes are a class of enzyme that can causes specific breaks in a DNA molecule.The mechanism by which restriction enzymes cut DNA is by causing hydrolysis of the phosphodiester bonds between the nucleotides that make up DNA.
Restriction enzymes can be broadly split into two types:

  • Exonucleases cleave DNA from the ends of the DNA molecule. The action of exonucleases will remove nucleotides from the ends of DNA.
  • Endonucleases cleave within the interior of the DNA molecule. The action of endonucleases will cause fragmentation of DNA molecules.

Endonucleases

Some examples of restriction sites
Some examples of restriction sites

Endonucleases are essential for recombinant DNA technology because they allow specific genes to be cut out and released from within a DNA molecule. The specificity of endonucleases is determined by a specific sequence of nucleotides that they can recognise and cleave. This sequence of nucleotides is called the restriction site and will vary between different types of endonucleases.

The key features of the restriction sites are the sequence of nucleotides, sequence length and site of cleavage. In addition most of the restriction sites are palidromic, meaning that they read the same forwards and backwards. If this is the case then there is cleavage of both strands of DNA that make up the DNA molecule.

Once a DNA molecule has been cleaved two fragments will be resolved. Each of these fragments will contain an 'end' that is from the cleaved restriction site. These ends can vary, depending on the exact position of the bonds cleaved. In general there are two broad types of ends resolved from fragmentation:

  • Blunt ends where the two strands of DNA are the same length, and so there is no overhanging strand. This types of ends occur when the cleavage sites on both strands are directly opposite each other.
  • Sticky ends where one of the stands of DNA is longer and so overhangs the other. This type of end occurs when the cleavage sites on both strands are not opposite each other. The examples shown in the diagram produce this types of end.


DNA polymerase

Vectors and Plasmids

Gene Cloning

Gel Electrophoresis

Blotting Techniques

Western Blotting

Southern Blotting

Northern Blotting

PCR (Polymerase Chain Reaction)

DNA Sequencing

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