Genetic Engineering - Lecture Notes - Principles of Biology | BIOL 1020, Study notes of Biology

Download Genetic Engineering - Lecture Notes - Principles of Biology | BIOL 1020 and more Study notes Biology in PDF only on Docsity! BIOL 1020 – CHAPTER 20 LECTURE NOTES Chapter 20: Genetic Engineering I. General outline of genetic engineering A. DNA cleavage B. Production of recombinant DNA C. Cloning of the recombinant DNA D. Screening clones II. Manipulating DNA: DNA cleavage and Production of Recombinant DNA A. restriction enzymes: molecular scissors with a twist 1. restriction enzymes, also called restriction endonucleases, are enzymes that cut DNA molecules in specific places 2. restriction enzymes vary considerably  hundreds of different kinds of restriction enzymes are known (recognizing different DNA sequences)  recognized sequence length varies (most common are “4-base cutters” and “6-base cutters”)  placement of cut varies; some leave “sticky ends”, others “blunt ends”  most recognized sequences are palindromic  the sequence on one strand matches that of the complementary strand read in the opposite direction  thus 5'-AGCGCT-3' would have a complementary strand 3'-TCGCGA-5' or reading from 5' to 3', 5'- AGCGCT-3' 3. restriction enzymes are mostly from bacteria, and their natural role is to destroy DNA from invading viruses B. making recombinant DNA 1. restriction enzymes are used to cut up DNA of interest and a “vector” into which you want to place the DNA, making restriction fragments 2. particularly when sticky ends are involved, the target DNA restriction fragment can form basepairs with the vector 3. DNA ligase is then used to join the DNA strand backbones III. Cloning of recombinant DNA: using vectors A. cloning is the process of making many genetically identical cells from cell containing recombinant DNA 1. the gene piece introduced in the recombinant DNA is said to be the DNA that is cloned 2. recombinant DNA is introduced to cells by a vector; the vector is usually maintained in the altered cell line B. a vector is a means of delivering recombinant DNA to an organism 1. vectors must have a way of getting into the host organism (transformation) 2. vectors must have some way of being propagated  some types of vectors remain free but are copied and distributed in cell division  some type of vectors have the inserted DNA integrate all or in part with the host DNA 3. vector DNA sequence must be known enough so that restriction sites can be accurately predicted and used C. most commonly, vectors are either plasmids, viruses, or yeast artificial chromosomes (YACs) 1. plasmids as vectors  the most commonly used vectors today are plasmids  plasmids are small, circular DNA molecules with at least one replication origin  most bacterial cells contain several plasmids  some eukaryotic cells commonly have plasmids (such as the yeast Saccharomyces cerevisiae)  plasmids vary in what organisms can maintain them (largely based on the type of replication origin they carry)  most plasmids carry genes that are expressed, again with variations depending on the host cell 2. viruses as vectors  viruses infect cells with their DNA; recombinant DNA in a virus can thus be transferred into cells  some of this “transduction” occurs naturally, but genetic engineers control and exploit the process 3. yeast artificial chromosomes (YACs) as vectors  eukaryotes can support and maintain larger pieces of DNA as chromosomes  YACs have the required elements of chromosomes (centromere, telomeres) and can be used as vectors for large segments of recombinant DNA in some eukaryotes D. vectors typically include a selectable marker and a cloning site 1. selectable markers usually are a gene for a product that the host cell cannot make itself, such as an antibiotic resistance factor 2. the cloning site on a vector is engineered with many possible sites for restriction enzyme cutting, where foreign DNA can be inserted E. the piece of foreign DNA inserted at a cloning site is said to be cloned, and the combined foreign DNA + vector is called recombinant DNA 1 of 4 BIOL 1020 – CHAPTER 20 LECTURE NOTES IV. Screening A. often many clones are made with various DNA pieces inserted B. screening is used to find the DNA of interest; typically: 1. a selectable marker is used to ensure that the vector is present 2. a second type of selectable marker is tested to ensure that the vector contains inserted DNA (that is, make sure it is recombinant DNA) 3. cells from cell colonies that pass the screens to this point are used as sources for making large numbers of cells; DNA from these cells is then subjected to other treatments to help identify cell lines containing the DNA of interest (for example, the probing covered later in these notes) V. DNA libraries A. the first step in working with the DNA of a species is to break the whole genome into manageable bits for study; this is done by creating DNA libraries B. vectors serve as the “books” in a DNA library – each “book” has a different piece of inserted DNA C. two main types of libraries are genomic libraries and cDNA libraries D. genomic libraries 1. raw genomic DNA is broken into fragments  sometimes the breaking is done mechanically  sometimes the breaking is done with restriction enzymes  often a combination is used 2. the broken DNA pieces are put into vectors and then the vectors into host cells 3. cells lines are maintained for each library piece (often, the whole genome is represented multiple times in the library for completeness) 4. the cell lines are given unique identifiers, and DNA probing techniques (described later) can be used to determine what lines carry particular cloned DNA sequences E. cDNA (complementary DNA) libraries 1. a more refined approach than genomic libraries, this type of library is based mainly on the coding regions of DNA 2. mRNAs are isolated from a cell and converted into complementary DNA using the enzyme reverse transcriptase 3. the cDNA is then inserted into vectors and the library is made and maintained just like a genomic library 4. different types of cDNA libraries can be made, reflecting the conditions under which cells made the original mRNAs 5. again, DNA probing techniques are used to find which lines have a cDNA of interest VI. Techniques used to manipulate and study DNA before and after cloning include: PCR, DNA gel electrophoresis, probing, DNA sequencing, and RFLPs A. DNA sequence amplification: PCR (polymerase chain reaction) is used to get enough DNA to work with 1. DNA polymerase can build a DNA strand provided there is a template strand, a primer, and dNTPs (deoxyribonucleotides of each type: dATP, dCTP, dGTP, and dTTP)  denaturation: heating a DNA molecule will eventually denature (“melt”) the double strands into separate single strands, breaking the hydrogen bonds between A-T and C-G basepairs; this can provide potential template strands  annealing of primers: when the DNA cools, basepairs will reform; if small, specific DNA primers are added in excess compared to the amount of target (template) DNA molecules, the DNA primers will tend to bind to the target DNA strands and keep the original double helices from reforming  primer extension: then, DNA polymerases can add dNTPs to make a complementary DNA strands, starting at the 3’ ends of the primers  if you repeat this through a series of cycles, you will exponentially make new DNA strands that cover a specific region of DNA, defined by the specific primers used – a polymerase chain reaction, where each cycle essentially doubles the amount of your target DNA fragment 2. PCR works well only when the DNA polymerase used can withstand temperatures that melt DNA strands  such enzymes are found in organisms that grow under very hot conditions (such as thermophilic bacteria from hot springs)  these are called heat-stable DNA polymerases (the best known one is Taq polymerase) 3. typically, the PCR works like this:  DNA melting is done 94°C (just a bit under the boiling temperature of water) for a minute or less  DNA annealing is done at a temperature around 50°C for a minute or less, but this varies depending on the DNA primers used and their optimal annealing temperature – you want to avoid getting too cool, where some nonspecific annealing can occur  DNA synthesis (primer extension) is performed at the optimal temperature for the heat-stable DNA polymerase, usually 72°C; the time given to extension is roughly 1 min per kilobase of DNA in the final target size 2 of 4