DNA Technology

DNA technology Some important vocab: • Bacterium - contains “naked” circular DNA • Biotechnology - science of recombining genes • Ligase - an enzyme that glues DNA pieces together • Plasmid - extrachromosomal DNA loop; 10-20 genes • rDNA - “recombinant DNA” • Restriction Enzyme - cuts DNA at a specific base sequence every time it finds it • Vector - an instrument used to inject DNA into a target location Transformation • Bacterial cell wall contains glycoproteins – Cell wall is weakened by heat and/or chemicals • A piece of DNA is placed in with these “competent” cells – Some of the cells absorb DNA through their weakened walls. They are transformed. Transduction • Note: Viruses are NOT shown to scale • Virus attaches to bacterial cell wall. • Viral DNA is injected into the bacterium • If the viral DNA is absorbed into the chromosome or plasmid = transduction. 1 extra thing: Reverse Transcriptase Genetic info in retroviruses is a special case and has an alternate flow of information: from RNA to DNA ● made possible by reverse transcriptase = an enzyme that copies the viral RNA into DNA which integrates into the host genome and becomes transcribed & translated for the assembly of new viral progeny. Conjugation • Bacterial are either (+) or (-) for a specific plasmid • A mating bridge grows between the (+) and (-) . why do you think it’s called a mating bridge? Conjugation • The plasmid begins to replicate, sending a copy to the (-) bacterium. • If conjugation is completed, genes have been exchanged as in any sexual reproductive situation. Summary Horizontal acquisitions of genetic info (primarily in prokaryotes) via these 3 processes increases variation!!! Restriction Enzymes are used with Recombinant DNA • Restriction enzymes do not just hack away at the DNA molecule! – Each enzyme cuts only at specific places. – These places, known as restriction sites, allow molecular biologists to cut DNA into certain size chunks. The site where EcoRI cuts is ? Restriction Enzymes Here’s how it works in the lab: • A piece of DNA with the desired gene is located or produced. Restriction Enzymes • A plasmid is designed with the proper base sequence in it. • The appropriate restriction enzyme is added. Restriction Enzymes • The enzyme (HindIII) cuts both the DNA around the gene and the plasmid. Restriction Enzymes • The enzyme ligase (think of it as glue) is added. • If all goes well, the gene will be inserted into the plasmid. Restriction Enzymes • Not every plasmid “grabs” the gene. • Some genes glue back to the DNA and some plasmids glue back together. • Scientists transform bacteria with plasmids and grow them Restriction Enzymes • Since bacterial cell machinery will operate from any DNA instructions, the protein coded for in the added DNA will be made along with… the normal proteins the bacteria makes. • The new protein is chemically isolated from the bacterial proteins, purified, and tested. • The result is a "genetically engineered protein” from recombinant DNA (rDNA). Polymerase Chain Reaction (PCR) • Small amounts of DNA are amplified • This DNA can be analyzed with electrophoresis +; DNA fingerprinting. – Up to 1:100,000,000 odds of 2 DNA’s being identical. why is PCR considered ‘DNA Replication in a test tube?” Gel Electrophoresis • Takes advantage of the charge on the DNA molecules ! – (-) charge fragments move toward the (+) electrode and separate based on size, which creates the bands we see (right), unique to every person on Earth! Gel Electrophoresis Well # Contents 1 Victim 2 Crime Scene 3 Suspect 1 4 Suspect 2 5 Suspect 3 6 Suspect 4 Which lane(s) match?