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BIOL 1020
Anne- Marie Singh

BIOL 1020 – CHAPTER 20 LECTURE NOTES Chapter 20: DNA Technology and Genomics I. General outline of genetic engineering a. DNAcleavage 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 i. restriction enzymes, also called restriction endonucleases, are enzymes that cut DNAmolecules in specific places ii. restriction enzymes vary considerably 1. hundreds of different kinds of restriction enzymes are known (recognizing different DNAsequences) 2. recognized sequence length varies (most common are “4-base cutters” and “6-base cutters”) 3. placement of cut varies; some leave “sticky ends”, others “blunt ends” 4. most recognized sequences are palindromic a. the sequence on one strand matches that of the complementary strand read in the opposite direction b. thus 5'-AGCGCT-3' would have a complementary strand 3'-TCGCGA-5' or reading from 5' to 3', 5'-AGCGCT-3' iii.restriction enzymes are mostly from bacteria, and their natural role is to destroy DNAfrom invading viruses b. making recombinant DNA i. restriction enzymes are used to cut up DNAof interest and a “vector” into which you want to place the DNA, making restriction fragments ii. particularly when sticky ends are involved, the target DNArestriction fragment can form basepairs with the vector iii.DNA ligase is then used to join the DNAstrand backbones III. Cloning of recombinant DNA: using vectors a. cloning is the process of making many genetically identical cells from cell containing recombinant DNA i. the gene piece introduced in the recombinant DNAis said to be the DNAthat is cloned ii. recombinant DNAis 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 DNAto an organism i. vectors must have a way of getting into the host organism (transformation) ii. vectors must have some way of being propagated 1. some types of vectors remain free but are copied and distributed in cell division 2. some type of vectors have the inserted DNA integrate all or in part with the host DNA iii.vector DNAsequence 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) i. plasmids as vectors 1. the most commonly used vectors today are plasmids 2. plasmids are small, circular DNAmolecules with at least one replication origin a. most bacterial cells contain several plasmids b. some eukaryotic cells commonly have plasmids (such as the yeast Saccharomyces cerevisiae) 3. plasmids vary in what organisms can maintain them (largely based on the type of replication origin they carry) 4. most plasmids carry genes that are expressed, again with variations depending on the host cell ii. viruses as vectors 1. viruses infect cells with their DNA; recombinant DNAin a virus can thus be transferred into cells 2. some of this “transduction” occurs naturally, but genetic engineers control and exploit the process iii.yeast artificial chromosomes (YACs) as vectors 1. eukaryotes can support and maintain larger pieces of DNAas chromosomes 2. YACs have the required elements of chromosomes (centromere, telomeres) and can be used as vectors for large segments of recombinant DNAin some eukaryotes d. vectors typically include a selectable marker and a cloning site i. selectable markers usually are a gene for a product that the host cell cannot make, such as an antibiotic resistance factor ii. the cloning site on a vector is engineered with many possible sites for restriction enzyme cutting, where foreign DNAcan be inserted e. the piece of foreign DNAinserted at a cloning site is said to be cloned, and the combined foreign DNA+ vector is called recombinant DNA IV. Screening a. Often many clones are made with various DNApieces inserted b. Screening is used to find the DNAof interest; typically: i. a selectable marker is used to ensure that the vector is present ii. a second type of selectable marker is tested to ensure that the vector contains inserted DNA(that is, make sure it is recombinant DNA) iii. cells from cell colonies that pass the screens to this point are used as sources for making large numbers of cells; DNAfrom 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 DNAof a species is to break the whole genome into manageable bits for study; this is done by creating DNAlibraries b. vectors serve as the “books” in a DNAlibrary – each “book” has a different piece of inserted DNA c. two main types of libraries are genomic libraries and cDNA libraries d. genomic libraries i. raw genomic DNAis broken into fragments 1. sometimes the breaking is done mechanically 2. sometimes the breaking is done with restriction enzymes 3. often a combination is used ii.the broken DNApieces are put into vectors and then the vectors into host cells iii.cells lines are maintained for each library piece (often, the whole genome is represented multiple times in the library for completeness) iv. 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 i. a more refined approach than genomic libraries, this type of library is based mainly on the coding regions of DNA ii.mRNAs are isolated from a cell and converted into complementary DNAusing the enzyme reverse transcriptase iii.the cDNAis then inserted into vectors and the library is made and maintained just like a genomic library iv. different types of cDNAlibraries can be made, reflecting the conditions under which cells made the original mRNAs v. again, DNAprobing techniques are used to find which lines have a cDNAof interest VI. Techniques used to manipulate and study DNA before and after cloning include: PCR, DNA gel electrophoresis, probing, DNA sequencing, and RFLPs a. DNAsequence amplification: PCR (polymerase chain reaction) is used to get enough DNAto work with i. DNApolymerase can build a DNAstrand provided there is a template strand, a primer, and dNTPs (deoxyribonucleotides of each type: dATP, dCTP, dGTP, and dTTP) 1. denaturation: heating a DNAmolecule 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 2. annealing of primers: when the DNAcools, basepairs will reform; if small, specific DNA primers are added in excess compared to the amount of target (template) DNAmolecules, the DNAprimers will tend to bind to the target DNA strands and keep the original double helices from reforming 3. primer extension: then, DNApolymerases can add dNTPs to make a complementary DNA strands, starting at the 3’ends of the primers 4. if you repeat this through a series of cycles, you will exponentially make new DNAstrands that cover a specific r
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