Chapter 18 RECOMBINANT DNA TECHNOLOGY Outline 1 Basic Tools and Techniques 2 Gene Isolation Characterization and Transfer 3 Application of Genetic Engineering to Humans Animals and Agriculture 4 Bioethics and Safety Issues I Basic Tools and Techniques Recombinant DNA technology is based on the central dogma of molecular biology that DNA makes RNA makes protein and the fact that if you modify the gene you can modify the protein Recombinant DNA techniques are used to study molecular aspects of life and to apply the information in a constructive and ethical manner to benefit life through academic and commercial research Recombinant DNA technology refers to a set of techniques used to isolate recombine transfer and express genes or DNA for further study Like any other technology recombinant DNA work is dependent on certain basic tools such as enzymes and plasmids and certain techniques are essential A selected list of such tools and techniques is briefly described below A Common enzymes used in molecular biology 1 Restriction endonucleases are enzymes isolated from prokaryotes that can recognize a specific DNA sequence and cleave the DNA at that recognition site or another place There are different types of restriction endonucleases present in prokaryotic cells to protect them from invading viruses and foreign DNA Some are generic i e cut DNA nonspecifically and some are specific Among the specific restriction enzymes type I and type III recognize at one site and cleave at another place Additionally types I and III have methylase activity Type II restriction endonucleases are the most commonly used restriction enzymes without methylase activity 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 cut DNA with the sequence 5 GAATTC 3 This is a six base pair recognition enzyme Other enzymes can recognize four base pairs or longer Once the restriction enzyme cuts the DNA they may leave an overhang of four base 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 similar sticky ends based on their complementarities Such enzymes that cut DNA at specific sites are needed to create recombinant DNA molecules 2 DNA polymerase DNA polymerases are used to make DNA in vitro DNA synthesis is accomplished by providing a DNA template suitable primers oligonucleotides 15 to 30 base long DNA primer complementary to template dNTPs a DNA polymerase Mg and a suitable buffer with the optimum pH and ionic condition Commonly used DNA polymerases are obtained from E coli T7 bacteriophage or thermostable bacteria and used for various applications such as DNA synthesis DNA sequencing and polymerase chain reactions 3 DNA ligase As we saw in DNA replication DNA ligase can catalyze the covalent bonding of the 3 and 5 ends of two DNA strands This is used to connect two DNA strands having blunt ends or complementary sticky ends created by restriction enzymes to make a recombinant DNA molecule E g the T4 DNA ligase obtained from the T4 phage is one the most commonly used ligases 4 Reverse transcriptase RT Reverse transcriptase is an RNA dependent DNA polymerase i e it uses an RNA template to make complementary DNA cDNA This is used in cDNA library construction and to amplify DNA from RNA The commonly used RTs are isolated from viruses such as AMV an avian virus and MMLV a marine virus Some thermostable DNA polymerases also have reverse transcriptase activity under different salt conditions B Vectors and hosts Vectors are the DNA vehicles that can carry genes from one organism to another and allow it to replicate in a particular host E coli is one of the most commonly used host systems in recombinant DNA Others include yeast plant cells and animal cells maintained in cell cultures Some commonly used vectors are plasmid or phage based 1 Plasmid vectors Plasmids are double stranded circular DNA molecules with an origin of replication an antibiotic marker gene for selection and restrictions sites that are unique and that can be used to insert a DNA fragment to be cloned Cloning refers to the process of making multiple identical copies of a particular DNA fragment or a gene after making the recombinant DNA and introducing it into a suitable bacterial host transformation Plasmid vectors are used to clone genes of relatively small size 100 bp to 15 kbp and they are relatively less efficient than phage vectors However they are easier to handle and are more stable to maintain than phage vectors E g pBluescript is one of the commonly used plasmid vectors 2 Phage vectors These are derived from the lambda phage a small phage approximately 48 kbp that can accommodate DNA fragments of 10 to 20 kbp in a region that is nonessential The nonessential region in the middle of the phage is removed and foreign 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 more efficient than transformation C Basic techniques 1 Gel electrophoresis This 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 are used 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 DNA in DNA sequencing gels depending upon the thickness length and size of the molecules being separated 2 Restriction fragment length polymorphism RFLP This is a technique commonly used to identify differences in the restriction pattern of a specific gene or DNA region between several individuals of a species or several related species The DNA is cut by selected restriction enzymes and fractionated on an agarose gel The differences in the sizes of the fragments obtained from the restriction digestion are used to identify the relationship between individuals For