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gene technology biology 1 lecture 14 • The importance of plasmids and restriction endonucleases • The 4 steps of genetic engineering • Screening • PCR and sequencing • Commercial applications of genetic engineering The first artificially created genome used a bacterium... • Bacteria have developed a series of enzymes, restriction endonucleases, to combat bateriophage viruses • Restriction enzymes cut DNA into strands at definitive points deemed by specific nucleotide sequences • Such nucleotides sequences are typically palindromes, e.g., G|AATTC CTTAA|G • Restriction enzymes cut at the same point: since the ends created are compatible, ‘sticky ends’, they can re-associate • plasmids, circular DNA strands found in prokaryotes, can be similarly digested by restriction enzymes, and can be used as vectors • DNA strands can be spliced into the plasmid, and the plasmid re-inserted into a bacterium • As the bacteria divides, the spliced DNA becomes part of the genome and is also replicated • Certain viruses can also be used as vectors The 4 steps of genetic engineering • • • • DNA cleavage and isolation Production of recombinant DNA Cloning Screening DNA cleavage and isolation • Use restriction endonucleases to break up DNA strands • Separate strands by electrophoresis (shorter fragments travel further when subjected to electric current) • Excise electrophoresis bands, redissolve, and use for stage 2 Production of recombinant DNA • Insert DNA fragments into plasmids or similar vectors also digested by same restriction enzymes (that is, same sticky ends) • Use vector to ‘infect’ host bacteria (e.g., E. coli) Cloning • Incubate infected bacterium, which divides exponentially • To help identification of correctly engineered recombinant DNA, choose a DNA strand that has a characteristic phenotype e.g., – Resistance to penicillin ampr – Ability to metabolize X-gal (z-gene) • Arrange recombination to preserve ampr, but interfere with z-gene Screening • Screen for correct phenotype • Use of probes to identify specific nucleotide sequences • Probes can be radioactively labeled: when the nucleic acid sequence of the probe links with the DNA sequence of interest, a radioactive recombinant is formed that can be detected Other techniques • PCR: polymerase chain reaction. Sequences of DNA are amplified using repeated sequences of denaturation, addition of primers, and primer extension • Southern blotting is used to identify DNA strands using a radioactively labeled probe • DNA fingerprinting analyzes restriction fragment length polymorphisms (RFLPs): point mutations specific to individuals causes different ‘banding’ in an electrophoresis gel • DNA can be sequenced using the Sanger method. Primers (deoxynucleotides and dioxynucleotides) are added to DNA fragments. Depending on nucleotide sequence, primers recombine with DNA in specific sequence Applications of genetic technology • • • • Pharmaceuticals Gene therapy Piggyback vaccines Agricultural applications – – – – Herbicide resistance Nitrogen fixation Insect resistance Farm animals (growth hormones) • Cloning