Ch 18 Recombinant DNA Technology 12 06 2014 12 06 2014 18 Recombinant DNA Technology 12 06 2014 Recombinant DNA technology allows genes conferring specific traits to be transferred from one organism to another w o species barrier Applications of recombinant DNA span from agriculture to modern medicine and forensics I Basic Tools and Techniques Recombinant DNA technology is based on the central dogma of molecular bio 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 info in a constructive and ethical manner to benefit life through academic and commercial research Recombinant DNA technology set of techniques used to isolate recombine transfer and express genes or DNA for further study Recombinant DNA work is dependent on certain basic tools A Common enzymes used in molecular biology Restriction endonucleases enzymes isolated from prokaryotes that recognize a specific DNA sequence and cleave the DNA at that recognition site or another place o Different types of restriction endonucleases protect cells from invading viruses and foreign DNA o Type I and type III recognize at one site and cleave at another place o Type II are the most commonly used enzymes w o methylase activity and they recognize and cleave at a particular DNA sequence o Enzymes are named after the bacterium they are isolated from o Other enzymes recognize four base pairs or longer o Once the 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 o Sticky ends 5 or 3 overhangs they can anneal w similar sticky ends based on their complementarity used to create recombinant DNA molecules DNA polymerase used to make DNA in vitro o DNA synthesis is accomplished by providing a DNA template suitable primers dNTPs a DNA polymerase Mg and a suitable buffer w the optimum pH and ionic condition DNA ligase can catalyze the covalent bonding of the 3 and 5 ends of two DNA strands o Used to connect two DNA strands having blunt ends or complementary sticky ends created by restriction enzymes to make recombinant DNA molecule Reverse transcriptase an RNA dependent DNA polymerase o It uses an RNA to make a complementary DNA cDNA o This is used in cDNA library construction and to amplify DNA from RNA kbp B Vectors and hosts Vectors the DNA vehicles that can carry genes from one organism to another and allow it to replicate in a particular host Plasmid vectors used to clone genes of relatively small size 100 bp to 15 o Cloning 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 o Relatively less efficient than phage vectors o Easier to handle and more stable to maintain than phage vectors Phage vectors derived from lambda phage small phage approx 48 kbp that can accommodate DNA fragments of 10 to 20 kbp in a region that is noessential o Nonessential region in the middle of the phage is removed and foreign DNA is inserted o The phage is used to infect the E coli host transfection to introduce the gene and to multiply it o Transfection is more efficient than transformation C Basic Techniques Gel electrophoresis used to fractionate DNA or RNA fragments based on their size o Negative charges on the DNA or RNA make them migrate toward the anode through tiny pores in agarose or polyacrylamide gel o Molecule migrate depending on their size and the electric voltage in the system o Larger molecules move slowly and smaller ones move quickly o The higher the voltage the faster they move o Agarose gels are used to fractionate DNA or RNA Easier to make but eh size fractionation is approximate o Polyacrylamide gels are used to fractionate proteins and DNA Relatively harder to make but provide better resolution of size Accuracy varies from approximate fractionation of proteins to accurate separation of DNA in DNA sequencing gels depending upon thickness length and size of the molecules being separated Restriction fragment length polymorphism RFLP commonly used to identify difference in the restriction pattern of a specific gene or DNA region between several individual of a special or several related species o DNA is cut by selected restriction enzymes and fractionated on agarose gel o Difference in the sizes of the fragments obtained from the restriction digestion are used to identify the relationship between individuals o Also referred to as DNA finger printing Radiolabeling DNA fragments to identify a DNA fragment in an organism label DNA w a radioisotope or fluorescent label that can be detected using X ray film Most commonly used isotopes are 35S and 32P to label DNA or RNA Used in rxns to synthesize DNA or RNA and are incorporated into strands during such synthesis Radiolabeling is used in DNA sequencing Southern and Northern DNA sequencing dideoxy chain termination method was developed by techniques Sanger DNA sequencing is done by doing an in vitro DNA synthesis using a DNA template a specific primer dNTPs and DNA polymerase to extend the new strand of DNA from the primer in a suitable buffer Sequence of complementary strand is obtained by stopping the chain extension by randomly incorporating dideoxynucleotides labeled w different color labels DNA fragments of random length are size fractionated by polyacrylamide electrophoresis and the sequence is determined by the fluorescent label attached to each dideoxy nucleotide in an automated sequencing method Alternate method using chemicals to sequence DNA was developed by Maxim and Gilbert Nextgen Sequencing modern high throughput sequencing methods Polymerase chain reaction PCR used to amplify large amounts of DNA from small amounts of samples Used extensively in cloning new genes from small amounts of RNA or DNA and in forensics to identify a suspect by amplifying the DNA found in samples at the crime scene In vitro DNA synthesis rxn two primers are used to synthesize DNA from a few copies of template using a thermostable DNA polymerase Includes a denaturation step an annealing step and an extension step Denaturation is done at 90C separates the dsDNA template The ssDNA anneals w the primer during the annealing step 55 70C Annealed primer is extended by the DNA polymerase incorporating dNTPs during the extension step These processes are done 32 times doubling the product each