4Discovery of restriction enzymes Bacteriophages infect bacteria by inserting their genetic material (DNA) into the DNA of bacteriaIn the 1960s, microbiologists discovered that some bacteria are protected from bacteriophage infection because they can restrict phage replicationScientists proposed that restricted growth of phages occurred cause the bacteria contained enzymes that could cut viral DNA into small pieces and prevent replicationThese enzymes were called restriction enzymes
5Restriction Enzymes Primarily found in bacteria Given names based on genus and species names of bacteria from which they are isolatedCut DNA by cleaving the phosphodiester bond (in sugar-phosphate backbone) that joins nucleotides in DNA strandShow specificity for certain DNA substratesEnzymes bind to, cut (digest) DNA within specific sequences of bases called recognition sequence or restriction siteTypically recognize 4, 6, or 8 nucleotide sequences
6Restriction EnzymesRestriction enzyme EcoRI, named because it was discovered in Escherichia coli
7Restriction EnzymesSome restriction enzymes cut DNA to create DNA fragments with overhanging single-stranded ends called “sticky” or cohesive ends
9Some enzymes generate fragments with non-overhanging or blunt ends Restriction EnzymesSome enzymes generate fragments with non-overhanging or blunt ends
10Restriction EnzymesEnzymes that produce cohesive ends are often favored over blunt-end cutters because DNA fragments with cohesive ends can easily be joined together.DNA from any source, bacteria, humans, dogs, frogs, dinosaurs, and ancient human remains can be digested by a particular restriction enzyme as long as it has the restriction site for that enzyme!
12Restriction Enzymes DNA cutting enzymes Very specific Cuts double stranded DNATwo incisions to cut DNA at sugar-phosphate bond on each strandVery specificEnzymes recognize short nucleotide sequencesEnzymes cut the DNA at specific points within DNAFor example – if the restriction enzyme cuts between a C and G then5'GGCC3' 3'CCGG5'5'GG CC3' 3'CC GG5'5'GG CC3' 3'CC GG5'
13Restriction EnzymesEach restriction enzyme recognizes a specific restriction site, a short nucleotide sequenceSome restriction enzymes cut straight across the DNASome restriction enzymes cut at offset nucleotidesFor example -G A A T T CC T T A A GG A A T T CC T T A A GG AATCCTTAA G
14Restriction EnzymesSequences that are cut at offset points produce overhanging pieces of DNA called “sticky ends”Sticky endG AATCCTTAA G
19Recombinant DNA Technology Technique that allows DNA to be combined from different sources
20Recombinant DNA/Restriction Enzyme Example In the early 1970s, a group of scientists joined together DNA from E. coli chromosomes and DNA from a primate virusThey isolated the DNA from each organism and cut them into fragments with EcoRIThey added the E. coli and viral DNA fragments to a reaction tube and added DNA ligaseThey created a hybrid of the two DNA types, recombinant DNA
21Recombinant DNA Technology Plasmids are considered extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosomePlasmids can be used as vectors, pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA
22Recombinant DNA Technology Plasmids are considered extrachromosomal DNA because they are present in the bacterial cytoplasm in addition to the bacterial chromosomePlasmids can be used as vectors, pieces of DNA that can accept, carry, and replicate (clone) other pieces of DNA
24NIH and RAC Scientists were concerned about: with what might happen if recombinant bacteria were to leave the labif such bacteria could transfer their genes to other cellssurvival in other organisms including humansNational Institutes of Health (NIH) formed the Recombinant DNA Advisory Committee (RAC) to evaluate the risks of recombinant DNA technology and establishing guidelines
25TransformationTransformation – process for inserting foreign DNA into bacteriaTreat bacterial cells with calcium chloride solutionsAdd plasmid DNA to cells chilled on iceBriefly heat the cell and DNA mixture, plasmid DNA will enter the bacterial cellOnce inside bacteria, plasmids replicate and express their genes
27SelectionJoining of DNA fragments and plasmids by transformation can be inefficientSome plasmids will rejoin and recircularize and not bond with any foreign DNADuring transformation, many cells will not take up DNARecombinant bacteria, those transformed with a recombinant plasmid, must be separated from nontransformed bacteria as well as cells with plasmids without foreign DNAThis process is called selection because it is designed to identify (select for) recombinant bacteria and prevent the growth of nontransformed bacteria and bacteria without foreign DNA
30Polymerase Chain Reaction Technique for making copies or amplifying a specific sequence of DNA in a short period of time.
31PCR (Polymerase Chain Reaction) 1. Target DNA to be amplified is added to a thin-walled tube and mixed with deoxyribonucleotides (dATP, dCTP, dGTP, dTTP), buffer and DNA polymerase. Primers are added to the mixture; they are complimentary to nucleotides on opposite ends of the target DNA.2. Tube is placed in a thermal cycler (gene cycler). This will take the sample through a series of reactions called the PCR cycle.
32Thermal CyclerDenaturation – tube is heated to ~94-96ºC separating DNA into single strandsHybridization (annealing) – tube is cooled to ~60-65ºC allowing primers to bond to ends of DNAExtension (elongation) – temperature raised slightly and DNA polymerase copies the DNA by binding to primer and making a new strandDNA polymerase used in this reaction is Taq DNA polymerase from Thermus aquaticcus. Bacteria is adapted to live in hot springs it has enzymes that can withstand high temperatures necessary for PCR.
34PCR At the end of one cycle, amount of DNA has doubled. Researchers can repeat cycle (usually times) to amplify millions of copies of DNA
35Uses of PCR Studying gene expression Detection of viral or bacterial infectionsDiagnosing genetic conditionsAmplifying trace amounts found at crime scenesAmplifying trace amounts found in ancient DNA
37Gel ElectrophoresisUsed to separate and visualize DNA fragments based on sizeAgarose gel is a semisolid material with small pores through which DNA can travel; fragments are separated by size – smaller fragments can travel further through pores than larger fragments
38Gel Electrophoresis1. To run a gel, it is submerged in buffer solution that will conduct electricity2. DNA samples are loaded into small wells and an electric current is passed through the gel3. The sugar-phosphate backbone renders DNA with a negative charge; therefore DNA will move to the positively charged endBecause migration distance is inversely proportional to the size of the DNA fragment, large DNA fragments migrate short distances and small fragments migrate fasterDyes are added to monitor DNA migration