DNA
Introduction
Large pieces of DNA can be broken up into smaller fragments by bacterial enzymes known as restriction endonucleases (RE). They bind to specific nucleotide sequences within DNA, known as recognition sequences/sites and cleave the phosphate backbone at a point within this sequence. -------------------------------------------------
The recognition sequences are usually fairly short (4-8 base pairs) and have two characteristics: 1) They are PALINDROMIC i.e. the sequence on one of the DNA strands is repeated in reverse on the other e.g. 5'...GAATTC...3' 3'...CTTAAG...5' | 2) They are cleaved by REs in one of two ways, either producing blunt ended fragments, or overhangingfragments | |

Since there are only 4 bases in DNA, the probability that a particular cut site is found in a random piece of DNA can be quite high. This is particularly true if the site contains only two bases e.g C and G and the DNA has a higher number of G/C pairs than A/T pairs. In this situation more fragments will be produced.
Plasmids are circular pieces of DNA found in bacteria and are frequently used in laboratories to house "foreign" genes. The ability to selectively cut plasmids with REs is extremely useful in genetic engineering.
The aim of this experiment is to digest two plasmids, one normal (plasmid 1), one mutant (plasmid 2) and analyse the results to determine the nature of the mutation.
Digesting plasmid DNA with restriction endonucleases
The two plasmids will be separately digested with two restriction enzymes:
Apa I - an overhanging end cutter which recognises the G/C rich sequence GGGCCC
Dra I - a blunt end cutter which recognises the A/T rich sequence TTTAAA
The image shows the eppendorfs used for the digestion. There are two eppendorfs for each plasmid - one for each restriction enzyme. Each digest will