Nucleic Acids                                                                     Lecture 23 (11/10/21)
                                                                                            A.           Nucleotides                                                                                                          • Reading:   Ch9; 309-313
                                                                                            B.           Nucleic Acids                                                                                                                                              Ch8; 283-286
                                                                                            C. The 4 S’s
                                                                                                                  1. Size
                                                                                                                  2. Solubility
                                                                                                                  3. Shape                                                                                                    • Problems:  Ch8; 13
                                                                                                                               a.       A-DNA
                                                                                                                               b.       Z-DNA
                                                                                                                               c.       Topology
                                                                                                                                            i.  Packaging
                                                                                                                                            ii. Supercoiling                                                                  NEXT
                                                                                                                  4. Stability              iii. Topoisomerases
                                                                                                                               a.       Nucleotides                                                                           • Reading:   Ch25; 916-921, 930-940
                                                                                                                                            i.  Tautomers
                                                                                                                                            ii. Acid/base
                                                                                                                               b.       Nucleic Acids                                                                         • Problems:  Ch25; 2,3,5,6,7,9,10,13,14,
                                                                                                                                            i.  Chemistry
                                                                                                                                            ii. Denaturation                                                                                                                           15,16,17
                                                                                                                                            iii. Stability
                                                                                                                                            iv. Nucleases                                                                                                                         1. X
                                                                                            D. Structure of the Information                                                                                                                                                       2. X
                                                                                                                  1. Exceptions to flow                                                                                                                                           3. Transformation of hosts
                                                                                                                  2. Structure                                                                                                                                                    4. Selection of transformants
                                                                                                                  3. Levels of Control                                                                                                                                                      a.          Selectable marker/gene
                                                                                            E.           Recombinant DNA:  Biochemical Basis of Biotechnology                                                                                                                               b.          Distinguish empty plasmids
                                                                                                                                                                                                                                                                                                           i.   Loss of resistance
                                                                                                                   1. Restriction enzymes, DNA ligase                                                                                                                                                      ii.  Reporter gene
                                                                                                                   2. Vectors and Inserts to make recombinant DNA (rDNA)                                                                                                          5. Expression
                                                                                                                              a.         Inserts                                                                                                                                              a.        Special vectors
                                                                                                                                            i.   cDNA                                                                                                                                         b.        Fusion proteins
                                                                                                                                            ii.  Genomic                                                                                                                                                   i.   purification
                                                                                                                              b.         Vectors                                                                                                                                                           ii.  labeling
                                                                                                                                                                                                                                                                                  6. Site-directed mutagenesis
                                                                                                                                Recombinant DNA and 
                                                                                                                                                                 Biotechnology
                                                                                                                                                                                                                                                                                                                                                                                                                       1
                 Recombinant DNA and Biotechnology 
                   Recombinant DNAis DNA made in the laboratory that is 
                   derived from at least two genetic sources.
                   Recombinant DNAhas allowed molecular biology to come 
                   full circle.
                                   FUNCTION
                         Biochemistry         Genetics
                          PROTEIN              GENE
                                    recDNA
                   Recombinant DNAhas one simple goal:  MAKE MORE
                 Recombinant DNA and Biotechnology 
                             Recombinant Proteins
                                ………coronavirus
                                                                          2
               Recombinant DNA and Biotechnology 
                  Recombinant DNAis DNA made in the laboratory that is 
                  derived from at least two genetic sources.
                •Biochemical Basis of Biotechnology
                  - Restriction enzymes, DNA ligase
                  - Vectors and Inserts to make recombinant DNA 
                   (rDNA)
                  - Transformation of hosts
                  - Selection of transformants
                  - Expression
                  - Site-directed mutagenesis
               Recombinant DNA and Biotechnology 
                                   Restriction enzymes are used to 
                                    cut DNA into fragments, which then 
                                    are spliced together in new 
                                    combinations.
                                   DNA ligase catalyzes the joining of 
                                    DNA fragments.
                                   Others you will see how they are 
                                    used when we go through the 
                                    processes.
                                                                 3
               Recombinant DNA and Biotechnology 
                            Restriction Sites
               Restriction enzymes recognize palindromic DNA sequences:
                  5ʼ…….GAATTC……3ʼ
                  3ʼ…….CTTAAG……5ʼ
               Some make straight cuts, others make staggered cuts, resulting in 
                overhangs or sticky ends.
                EXAMPLES:
               Recombinant DNA and Biotechnology 
                            Restriction Sites
               Restriction enzymes recognize palindromic DNA sequences:
                  5ʼ…….GAATTC……3ʼ
                  5ʼ…….G-3ʼ            5ʼ-AATTC……3ʼ
                  3ʼ…….CTTAAG……5ʼ
                  3ʼ…….CTTAA-5ʼ           3ʼ-G……5ʼ
               Some make straight cuts, others make staggered cuts, resulting in 
                overhangs or sticky ends.
                EXAMPLES:
                                                                 4