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UT BIO 311C - Chapter 18: RECOMBINANT DNA TECHNOLOGY

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I. Basic Tools and TechniquesII. Gene Isolation, Characterization, and TransferIII. Applications of Recombinant DNA TechnologyIV. Safety and EthicsChapter 18: RECOMBINANT DNATECHNOLOGYOutline1. Basic Tools and Techniques2. Gene Isolation, Characterization, and Transfer3. Application of Genetic Engineering to Humans, Animals, and Agriculture4. Bioethics and Safety IssuesI. Basic Tools and TechniquesRecombinant DNA technology is based on the central dogma ofmolecular biology that DNA makes RNA makes protein and the fact that ifyou modify the gene you can modify the protein. Recombinant DNAtechniques are used to study molecular aspects of life and to apply theinformation in a constructive and ethical manner to benefit life throughacademic and commercial research. Recombinant DNA technology refersto a set of techniques used to isolate, recombine, transfer, and expressgenes or DNA for further study. Like any other technology, recombinantDNA work is dependent on certain basic tools such as enzymes andplasmids, and certain techniques are essential. A selected list of suchtools and techniques is briefly described below.A. Common enzymes used in molecular biology1. Restriction endonucleasesare enzymes isolated from prokaryotes thatcan recognize a specific DNA sequence and cleave the DNA at thatrecognition site or another place. There are different types of restrictionendonucleases present in prokaryotic cells to protect them from invadingviruses and foreign DNA. Some are generic, i.e., cut DNA nonspecifically,and some are specific. Among the specific restriction enzymes, type I andtype III recognize at one site and cleave at another place. Additionally,types I and III have methylase activity. Type II restriction endonucleasesare the most commonly used restriction enzymes without methylaseactivity, and they recognize and cleave at a particular DNA sequence.The enzymes are named after the bacterium they are isolated from. E.g.,the enzyme EcoRI, isolated fromEscherichia coli,can recognize and cutDNA with the sequence 5’-GAATTC-3'. This is a six-base pair recognitionenzyme. Other enzymes can recognize four base pairs or longer. Oncethe restriction enzyme cuts the DNA, they may leave an overhang of fourbase pairs at the 5' or 3' end or they may leave a blunt end. The 5' or 3'overhangs are called sticky ends because they can anneal with similarsticky ends based on their complementarities. Such enzymes that cutDNA at specific sites are needed to create recombinant DNA molecules.2. DNA polymerase:DNA polymerases are used to makeDNA in vitro. DNA synthesis is accomplished by providinga DNA template, suitable primers (oligonucleotides, 15 to30 base long, DNA primer complementary to template),dNTPs, a DNA polymerase, Mg++, and a suitable bufferwith the optimum pH and ionic condition. Commonly usedDNA polymerases are obtained fromE. coli,T7bacteriophage, or thermostable bacteria and used forvarious applications, such as DNA synthesis, DNAsequencing, and polymerase chain reactions.3. DNA ligase:As we saw in DNA replication, DNA ligasecan catalyze the covalent bonding of the 3' and 5' ends oftwo DNA strands. This is used to connect two DNA strandshaving blunt ends or complementary sticky ends createdby restriction enzymes to make a recombinant DNAmolecule. E.g., the T4 DNA ligase obtained from the T4phage is one the most commonly used ligases.4. Reverse transcriptase(RT): Reverse transcriptase is anRNA-dependent DNA polymerase, i.e., it uses an RNAtemplate to make complementary DNA (cDNA). This isused in cDNA library construction and to amplify DNA fromRNA. The commonly used RTs are isolated from virusessuch as AMV (an avian virus) and MMLV (a marine virus).Some thermostable DNA polymerases also have reversetranscriptase activity under different salt conditions.B. Vectors and hostsVectors are the DNA vehicles that can carry genes from one organism toanother, and allow it to replicate in a particular host.E. coliis one of themost commonly used host systems in recombinant DNA. Others includeyeast, plant cells, and animal cells maintained in cell cultures. Somecommonly used vectors are plasmid or phage-based.1. Plasmid vectors:Plasmids are double-stranded circular DNA moleculeswith an origin of replication, an antibiotic marker gene for selection, andrestrictions sites that are unique and that can be used to insert a DNAfragment to be cloned. Cloning refers to the process of making multipleidentical copies of a particular DNA fragment or a gene after making therecombinant DNA and introducing it into a suitable bacterial host(transformation). Plasmid vectors are used to clone genes of relativelysmall size (100 bp to 15 kbp) and they are relatively less efficient thanphage vectors. However, they are easier to handle and are more stable tomaintain than phage vectors. E.g., pBluescript is one of the commonlyused plasmid vectors.2. Phage vectors:These are derived from the lambda phage, a smallphage (approximately 48 kbp) that can accommodate DNA fragments of10 to 20 kbp in a region that is nonessential.The nonessential region in the middle of the phage is removed andforeign DNA is inserted. Then the phage is used to infect theE. colihost(transfection) to introduce the gene and to multiply it. Transfection is moreefficient than transformation.C. Basic techniques1. Gel electrophoresisThis is used to fractionate DNA or RNA fragments based on their size. The negative charges on the DNA or RNA make them migrate toward the anode (+) through tiny pores in the agarose or polyacrylamide gel. The molecules migrate depending on their size and electric voltage in the system. Larger molecules move slowly and smaller molecules move quickly. The higher the voltage, the faster they move. Agarose gels are used to fractionate DNA or RNA. These are easier to make, but the size fractionation is approximate. Polyacrylamide gels areused to fractionate proteins and DNA. Polyacrylamide gels are relatively harder to make but provide better resolution of size. The accuracy of polyacrylamide gels varies from approximate fractionation of proteins to accurate separation of


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