2 Manipulating DNAScientists use their knowledge of the structure of DNA and its chemical properties to study and change DNA moleculesDifferent techniques are used to study and change DNA moleculesGenetic Engineering – making changes in the DNA code of a living organismBacteria are the workhorses of modern biotechnology
3 Tools of Molecular Biology: Recombinant DNA Recombinant DNA techniques can help biologists produce large quantities of a desired proteinTo work with genes in the laboratory, biologists often use bacterial plasmids, small, circular DNA moleculesPlasmids can:can carry virtually any gene,can act as vectors, DNA carriers that move genes from one cell to another, andare ideal for gene cloning, the production of multiple identical copies of a gene-carrying piece of DNA.
4 Recombinant DNARecombinant DNA is produced by combining two ingredients:a bacterial plasmid andthe gene of interest.To combine these ingredients, a piece of DNA must be spliced into a plasmid
5 Creating Recombinant DNA In order to create Recombinant DNA, there needs to be:DNA extractionCells opened to separate DNA from other cell partsCutting DNADNA too large to study, so biologists “cut” them into smaller fragments using restriction enzymes. Many restriction enzymes are known and each one cuts DNA at a specific sequence of nucleotidesProduces pieces of DNA called restriction fragments with “sticky ends” important for joining DNA from different sources.Splicing DNA back togetherDNA ligase connects the DNA pieces into continuous strands by forming bonds between adjacent nucleotides
6 Restriction Enzymes Recognition site (recognition sequence) for a restriction enzymeRestriction Enzymes1A restriction enzyme cuts the DNA into fragments.RestrictionenzymeSticky endSticky end
7 Restriction Enzymes Recognition site (recognition sequence) for a restriction enzymeRestriction EnzymesDNA1A restriction enzyme cuts the DNA into fragments.RestrictionenzymeSticky endSticky end2A DNA fragment is added from another source.
8 Restriction Enzymes Recognition site (recognition sequence) for a restriction enzymeRestriction EnzymesDNA1A restriction enzyme cuts the DNA into fragments.RestrictionenzymeSticky endSticky end2A DNA fragment is added from another source.3Fragments stick together bybase pairing.
9 Restriction Enzymes Recognition site (recognition sequence) for a restriction enzymeRestriction EnzymesDNA1A restriction enzyme cuts the DNA into fragments.RestrictionenzymeSticky endSticky end2A DNA fragment is added from another source.3Fragments stick together bybase pairing.4DNA ligase joins the fragments into strands.DNAligaseRecombinant DNA molecule
10 Recognition sequences Restriction EnzymesRecognition sequencesDNA sequenceRestriction enzyme EcoRI cuts the DNA into fragments.Sticky end
11 Recognition sequences Restriction EnzymesRecognition sequencesDNA sequenceRestriction enzyme EcoRI cuts the DNA into fragments.Sticky end
12 Finding the Gene of Interest How can a researcher obtain DNA that encodes a particular gene of interest? First, you have to have an idea of what the gene is you want to work with (get a genomic library). Then:Using a nucleic acid probe consisting of a short single strand of DNA with a complementary sequence and labeled with either a radioactive isotope or a fluorescent dye.Or by synthesizing it through reverse transcriptase (viral enzyme that makes DNA)Or by making it by scratch with machines
13 DNA Profiling and Forensic Science can be used to determine if two samples of genetic material are from a particular individual andhas rapidly revolutionized the field of forensics, the scientific analysis of evidence from crime scenes.To produce a DNA profile, scientists compare sequences in the genome that vary from person to person.
14 DNA Profiling and Forensic Science DNA profiling can be used totest the guilt of suspected criminals,identify tissue samples of victims,resolve paternity cases,identify contraband animal products, andtrace the evolutionary history of organisms.
15 DNA Profiling Techniques: Making Copies of DNA Polymerase Chain Reaction (PCR) techniqueAllows biologists to make many copies of a specific piece of DNADNA strands separated with heat, then cooled to allow DNA Polymerase to start making new copies of DNAA few dozen heat and cool cycles results in many copies of DNA
16 Making Copies of DNA DNA polymerase adds complementary strand DNA heated to separate strandsDNA fragment to be copiedPCR cycles 1DNA copies 1234485 etc.16 etc.
17 DNA Profiling Techniques: STR Analysis Short tandem repeats (STRs) are:short sequences of DNA that are repeated many times, tandemly (one after another), in the genome.STR analysisProves two samples come from the same person - Everyone has these repetitive DNA sequences, but in different lengths and a different number of themcompares the lengths of STR sequences at specific sites in the genomeuses gel electrophoresis, a method for sorting DNA by size
18 DNA Profiling Techniques: Gel Electrophoresis Used to separate DNA fragments. DNA fragments placed in a gel and electricity is applied to the gel. DNA molecules are negatively charged and move towards the positive end of the gel. Smaller DNA fragments move faster and fartherThis technique used to compare the genomes of different organisms or even different people
19 Gel Electrophoresis Power source DNA plus restriction enzyme Longer fragmentsShorter fragmentsMixture of DNA fragmentsGel
21 Blood found at Crime Scene Gel ElectrophoresisWhich suspect should have more questioning?DNA from VictimDNA from Suspect #1DNA from Suspect #2DNA from Suspect #3DNA from Suspect #4Blood found at Crime Scene
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