Restriction Enzymes (also known as restriction endonucleases) What are restriction enzymes? • An enzyme that cuts DNA at specific nucleotide sequences known as restriction sites • Naturally found in bacteria and have evolved to provide a defense mechanism against invading viruses • In bacteria, restriction enzymes selectively cut up foreign DNA in a process called restriction Restriction Enzymes in Bacteria Restriction Enzymes • Cut both strands of the DNA double helix • Over 3000 enzymes have been identified • More than 600 available commercially • Routinely used for DNA modification and manipulation in laboratories (“molecular scissors”) Restriction Sites • Also known as recognition sites • Generally genetic palindromic sequences • A palindromic sequence in DNA one in which the 5’ to 3’ base pair sequence is identical on both strands • Usually a 4 or 6 base pair sequence Restriction Sites • The enzymes scan DNA sequences, find a very specific set of nucleotides and make a cut Hae III • HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases - GGCC 5’ TGACGGGTTCGAGGCCAG 3’ 3’ ACTGCCCAAGGTCCGGTC 5’ Hae III • Once the recognition site was found HaeIII will go to work cutting (cleaving) the DNA Blunt Ends versus Sticky Ends • Hae III produces “blunt ends” when cleaving DNA • Other enzymes produce “sticky ends” blunt end sticky end Restriction Enzyme Names • Named after the type of bacteria in which the enzyme is found and the order in which the restriction enzyme was identified and isolated EcoRI for example R strain of E.coli bacteria I as it is was the first E. coli restriction enzyme to be discovered. Restriction Enzymes and Gene Cloning Separating Restriction Fragments • Restriction enzymes are used to generate RFLPs (“rif-lips”) - Restriction fragment length polymorphisms • RFLPs are differences among individuals in the lengths of DNA fragments cut by enzymes • RFLPs can be separated using electrophoresis and then analyzed Generation of RFLPs Separating Restriction Fragments Gel Electrophoresis Applications Gel Electrophoresis Applications • DNA sequencing (old school method) • Forensic purposes • Paternity testing • Examine evolutionary relationships among organisms (similarities and differences) • Diagnose genetic disorders or gene testing • Protein analysis • Determination of impurities in a sample DNA Sequencing • Sanger Method - DNA sequencing using gel electrophoresis (method for ~25 years) • Uses radioactively labeled dideoxyribonucleotides DNA Sequencing DNA Fingerprinting • A test used to identify and evaluate genetic information DNA Fingerprinting Steps Forensics • Restriction enzymes and gel electrophoresis are used to create DNA fingerprints to analyze DNA samples from crime scenes Paternity Testing • The pattern of DNA fragments (produced by the restriction enzyme digest) is compared and if the child's DNA looks like a combination of the two parents' DNA, then the child is theirs Evolutionary Relationships • Which of the species, X or Y, is more closely related to species Z? Diagnose Genetic Disease • Molecular diagnosis of a particular inherited disease .