GEN 3022 1st Edition Lecture 32Outline of Last Lecture I. Gene conversiona. Example Ib. Gap repair synthesis vs. DNA mismatch repairII. Transpositiona. Cut-and-paste mechanismb. Reverse transcriptaseIII. Homologous Recombination a. Holliday modelb. Recombinant and nonrecombinant chromosomesc. Heteroduplex DNAOutline of Current LectureI. Recombinant DNA technologya. Discoveryb. Importance of recombinant DNA technologyII. Gene cloning a. Cloning experimentsi. cDNA ii. vector DNAiii. preparation of DNAiv. host cellb. restriction enzymesIII. Steps in gene cloning (of human B-globin gene)a. Plasmid: AmpR and lacZb. cDNAc. Restriction enzymesd. Recombinant evente. Testing for the desired genef. Net Result of cloningIV. Polymerase Chain Reactiona. Starting materialsi. Template DNAii. Oligonucleotide primersThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.iii. Deoxynucleoside triphosphatesiv. Taq polymeraseb. Processc. Resultd. Reverse transcriptase PCRCurrent LectureI. Recombinant DNA technology: the use of in vitro molecular techniques to isolate andmanipulate fragments of DNAa. Discovery: discovered in early 1970s by researchers at Stanford who were trying to construct chimeric molecules called recombinant DNA molecules. This led to introducing the molecules into living cells where they are replicated to produce many copies.b. Importance of recombinant DNA technology: Recombinant DNA technology opened the door to gene cloning research, both of which have been critical to understanding gene structure and function.II. Gene cloning: the technique of isolating and making many copies of a gene.a. Cloning experiments: involve two kinds of molecules- cDNA (or chromosomal DNA) and vector DNAi. Preparation of chromosomal DNA or cDNA: cellular tissue is obtained from organism of interest, the cells are lysed (broken open), and the DNA is extracted and purified. ii. Host cell: cell that harbors the vector. When a vector is replicated inside a host cell, the DNA that it carries is also replicated.b. cDNA: also known as complementary DNAc. Vectors: serves as the carrier for the DNA segment that is to be cloned. Can replicate independently of the host chromosomal DNA. These vectors were originally derived from plasmids and viruses. d. Restriction enzymes: also called restriction endonucleases. Bind to specific sequences (usually palindromic sequences) in the DNA and cleave strands at two defined locations (one on each strand) to create “sticky ends”. i. The ends will hydrogen bond to each other due to their complementary sequences. Some generate blunt ends though. ii. Made naturally by many types of bacteria (protect bacterial cells from invasion by foreign DNA, especially bacteriophages). III. Steps in gene cloning (of human B-globin gene)a. Plasmid: AmpR and lacZ. AmpR confers antibiotic resistance to the host cell. Therefore, only cells that have taken up the vector will grow on an ampicillinplate. LacZ encodes for B-galactosidase and provides a means to determine which cells grew on the ampicillin plate and contain the cloned gene. This is called screening.b. cDNA: contains desired gene. Combined with vector DNA and restriction enzymes. Formed using reverse transcriptase and can be single or double stranded. Important advantage is the lack of introns, which allows researchers to focus their attention on the coding sequence of a gene and the expression of the encoded protein. c. Restriction enzymes: cut up cDNA and plasmid DNA (vector) at specific sites.d. Recombinant event: the plasmid DNA and cDNA combine to form recombinant vectors. Only some of these contain the desired gene. e. Testing for the desired gene: the vectors are inserted into host cells where they replicate. This process is called transformation. The cells are then allowed to grow on an ampicillin plate. Cells that contain functional B-galactosidase do not contain the desired gene and will turn blue. The cells that remain white contain the desired recombinant vector. f. Net Result of cloning: goal is to produce an enormous number of copies of a gene. Even though a single bacterial cell takes up just one copy of a vector duringtransformation, the cloned gene is amplified when the vector is replicated in the host cell or when the bacterial cell itself divides (about every 20 minutes).IV. Polymerase Chain Reaction: a technique developed by Kary Mullis in 1985 to copy DNA without the aid of vectors and host cells. Can also be used to amplify chromosomal DNA (not just cDNA), but it is not as specific. This is what is used to amplify DNA samples found at crime scenes. a. Starting materialsi. Template DNA: contains the region that is amplified by PCR. ii. Oligonucleotide primers: complementary to sequences at the ends of the DNA fragment to be amplified. Synthetic and usually 15-20 nucleotides in length.iii. Deoxynucleoside triphosphates: provide the precursors for DNA synthesis. iv. Taq polymerase: DNA polymerase that is isolated from the bacterium Thermusaquaticus. Necessary because it is stable under high temperatures and PCR involves heating steps that would inactivate many other DNA polymerases.b. Process: carried out in a thermocycler that is programmed to maintain a certain range of temperatures and automates the timing of each cycle. Primers are selected that bind to either side of the desired gene. Many PCR cycles occur andeach cycle produces copies of the region of interest following the general patternof denaturing-annealing-synthesis.c. Result: a typical PCR is 20-30 cycles, which increases the DNA sequence to 220 fold of the original (assuming 100% efficiency).d. Reverse transcriptase PCR: used to clone and quantitate RNA in living cells. RNA is first isolated from the sample and mixed with reverse transcriptase and a primer that will anneal to the 3’ end of the RNA of interest. This generates a single-stranded cDNA which can be used as a template DNA in conventional PCR. RT-PCR is very sensitive and can detect the expression of small amounts of RNA in a single