4/26/2015. Cut DNA either: Cut DNA either:
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1 Ch.20 Enzymes that cut DNA at specific sequences (restriction sites) resulting in segments of DNA (restriction fragments) Typically 4-8 bp in length & often palindromic Isolated from bacteria (Hundreds have been found) Normally function by cutting up foreign DNA Cut DNA either: In middle of sequence (blunt ends) Cut DNA either: In middle of sequence (blunt ends) Staggered, producing short single-stranded overhangs (sticky ends) Different enzymes recognize and cut different restriction sites Most frequently used to create recombinant DNA (more on that in a bit ) DNA molecules able to carry foreign DNA into a host cell and replicate there Bacterial plasmids are most common (also phages, viruses, artificial chromosomes) 1
2 DNA molecules able to carry foreign DNA into a host cell and replicate there Typically contain: Origin of Replication Region containing multiple restriction sites Gene(s) enabling screening of cells having taken up the plasmid (usually antibiotic resistance ampicillin is common) Combining genetic information from two or more organisms to express new traits (transgenic organisms) Requires restriction enzymes and vectors Successfully accomplished in all domains Used in a variety of fields (medicine, agriculture, industry, research) Using reporter genes in vectors that enable detection of successful engineering Often utilize antibiotic resistance, GFP Frequency of success if usually quite low (and varies by size of plasmid being worked with) Collections of DNA sequences, stored in vectors Usually use BACs (Bacterial Artificial Chromosomes) Allow large fragments to be inserted, decreasing the number of necessary clones Often subcloned into normal plasmids later if individual fragments are required 2
3 Collections of DNA sequences, containing only transcribed genes from isolated mrna sequences Collections of DNA sequences, containing only transcribed genes from isolated mrna sequences Obtained using Reverse Transcriptase (from retroviruses) Collections of DNA sequences, containing only transcribed genes from isolated mrna sequences Allows prokaryotes to express eukaryotic genes Process separating fragments of DNA based on size DNA is (-) charged and always moves to the (+) Smaller fragments move faster Each restriction enzyme digest & DNA fragment shows unique banding pattern (DNA fingerprint) Thermocycler Process producing many copies of a specific DNA sequence without cells Instead of DNA helicase, PCR alternates between heating & cooling Requires Taq DNA Polymerase (heat-stable) Isolated from thermophilic bacteria (Yellowstone NP) 1. Denaturation Heating DNA to extremely high temps (94-98 C) DNA strands separate 3
4 2. Annealing Temp is lowered (50-65 C) Allows primers to bind to template strands 3. Extension/Elongation Temp raised slightly (72-80 C) Taq DNA Polymerase attaches to primer and replicates sequence Repeated over & over Each cycle doubles the number of DNA molecules (Most PCRs run for 30 cycles) 2 30 = over 1 billion!! Using biotech tools to look for presence/ absence of particular gene sequences Often require restriction enzymes, PCR, electrophoresis Historically analyze RFLPs (Restriction Fragment Length Polymorphisms) Other methods are often used today (look at expression) Using biotech tools to look for presence/ absence of particular gene sequences Genetic Profiles Analyze areas of genome with repeating sequences called short tandem repeats (STRs) Highly varying from person to person Only a few sites need to be tested to confirm or rule out (crime scene, paternity, etc) Using biotech tools to look for presence/ absence of particular gene sequences Health Issues Analyzing RFLPs to identify various traits β-globin allele loses a DdeI restriction site on the sickle-cell mutant allele 4
5 In Britain, approved for humans as a fertility treatment by Parliament in Feb Nucleus from a fertilized egg (with defective mitochondria) is removed and transferred to another egg Results in a healthy fertilized egg that is produced from three genetic parents (mom & dad nuclear DNA, donor mitochondrial DNA) Reproductive Cloning Process that produces a genetically identical offspring to the parent Reproductive Cloning Process that produces a genetically identical offspring to the parent Reproductive Cloning Process that produces a genetically identical offspring to the parent Therapeutic Cloning Process that produces specific cells to be used for medical purposes Requires pluripotent stem cells (embryonic or adult) Therapeutic Cloning Process that produces specific cells to be used for medical purposes Requires pluripotent stem cells (embryonic or adult) 5
6 Process that determines the sequence of bases in a segment of DNA DNA is replicated in presence of ddnts (dideoxyribonucleic acids) Incorporation of ddnt into strand of DNA terminates replication Process allowing visualization of gene expression at a genome-wide level mrna is reverse-transcribed to create cdna cdna is compared to known DNA fragments Any cdna that binds indicates those genes are being expressed by the cell p p.412 1,4 p.416 1,2,4 p.423 1,2 6
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