I. Terminology
     A. Recombinant DNA
     B. Clone
II. Type II restriction endonucleases cut DNA at specific sequence
     A. Usually recognize palindromes: GGATCC
     B. May not work on methylated DNA
     C. Naming: first letter of genus, first two letters of species
     D. Blunt end or sticky end cuts
     E. Used by bacteria to destroy bacteriophage DNA
          1. Bacterial DNA is methylated
          2. Viral DNA is not methylated
          3. When bacterial DNA replicates, old strand methylated
          4. Methylase recognizes methyl group on one strand, adds on opposite strand
     F. Restriction mapping – location of sites within DNA
          1. Separate and size fragments by electrophoresis
          2. Often label ends
          3. Double digests (fig. 12.30); used to produce overlapping pieces
III. Cloning vectors
     A. Plasmid
          1. Joining two DNAs adds extra restriction site, making it east to later remove added DNA
          2. Tailing, blunt-end ligation
          3. Add poly-dT to one DNA and poly-dA to other
          4. Blunt end – add ligase and form many different hybrids
          5. Linkers – attach to blunt end and make a new sticky end with enzyme
     B. Phage
     C. Shuttle vector
          1. Hybrid vectors used to move DNA form place to place
     D. Yeast artificial chromosomes (YACs)
     E. Bacterial artificial chromosomes (BACs)
     F. Eukaryotic vectors – used because of difference between prokaryotic and eukaryotic genes
          1. Yeast vectors – YACs (fig. 12.19)
               a. Yeast centromere and replicating sequence
               b. Can handle more than 500 kilobases (kb) of DNA
          2. Animal vectors – SV40, but with part of SV40 replaced
          3. Plant vectors – Ti plasmid; Arabidopsis thaliana – an ideal plant for gene studies
     G. Selectable marker
     H. Complementary DNA (cDNA)
IV. Cloning
     A. Plasmid cleavage – nonessential site of plasmid function; usually one site per plasmid
     B. Passenger DNA must be functional
     C. Charon phage
          1. DNA inserted into middle of lambda genome
          2. Can handle large pieces of DNA (15-24 Kb)
     D. Cosmids – plasmid DNA + cohesive ends + lambda capsids
V. Screening for correct clone - selectable marker
     A. Charon phages – ability to infect E. coli
     B. Plasmids – antibiotic resistance; insertion of DNA may eliminate antibiotic resistance
VI. Making library
     A. Genomic library
          1. Genomic DNA broken into small pieces
          2. Each fragment cloned
     B. Chromosomal library
          1. For mapping genes on a given chromosome
          2. Use of mouse/human hybrids
     C. cDNA library
          1. DNA to clone – only one gene
          2. Isolate mRNA; use reverse transcriptase to make cDNA (fig. 12.12)
          3. Synthesis – if amino acid sequence known, make DNA with help of genetic code
          4. Cut genome with enzymes
VII. Screening a library
     A. Probe
     B. Complementation of mutations
VIII. Finding a gene – need radioactive probe; cDNA often used
     A. Southern blotting – DNA fragments separated, transferred to filter mixed with probe
     B. Northern blotting – RNA separated, transferred, and probed
     C. Dot blotting – colonies lysed and hybridized
     D. Western blotting
          1. Looks for expression of protein
          2. Requires expression vector
          3. Probes are usually antibodies
     E. Chromosome walking – looks for overlapping regions, then identifies adjacent regions (fig 12.17)
     F. Chromosome jumping allows identification of ends of region without looking at middle
     G. Heteroduplex analysis – look for paired regions with electron microscope
IX. Eukaryotic expression
     A. Transfection – transfer foreign DNA to eukaryote
          1. Injection into oocyte; DNA incorporated into chromosome
          2. Retroviruses – part of genome replaced with passenger DNA (fig. 12.25)
          3. Knockout mice
                a. Oocyte transfected with defective gene which replaces normal gene
                b. Mice bred to produce homozygous defectives
     B. Electroporation  - electric current used to get DNA uptake
     C. Liposomes – DNA inside artificial membranes
     D. Biolistic – shooting microprojectiles
     E. Reporter systems – shows when a gene is expressed
          1. Luciferase: needs ATP to work
          2. Green fluorescent protein needs only UV light (fig. 12.27)
X. Various techniques
     A. DNA sequencing – dideoxy method
          1. Use 2' and 3; dideoxy sugars – new bases cannot be added to chain
          2. Four dideoxynucleotides stop DNA synthesis after each base (fig. 12.35, 12.36)
          3. Separate fragments
          4. Read sequence up from bottom of gel
          5. Yields sequence of complementary strand from that originally used
          6. Can use fluorescent dyes and lasers
     B. Polymerase chain reaction(PCR)
          1. Need small known sequence
          2. Many repeated cycles of DNA duplication
          3. Results in amplification of DNA sequence
     C. Restriction fragment length polymorphisms (RFLPs)
          1. Looks for alleles within region
          2. Use probes to small regions and get different size fragments from different people; alleles
          3. Variable number of tandem repeats (VNTR)
               a. Many bands almost give fingerprint of DNA
               b. May be used to identify individuals; must be careful of inclusion vs. exclusion
     D. Positional cloning
     E. DNA fingerprinting
XI. Human Genome Project
     A. Locate genes by using probes and chromosomal deletions
     B. Produce map based on RFLPs and overlapping contiguous clones
        1.  Requires many cosmids for DNA of one chromosome
     C. Locating breast cancer gene
          1. Try to correlate pedigrees and RFLP markers
          2. Long arm of chromosome 17
          3. Zinc-finger motif; 1863 amino acids; may function as tumor suppressor
     D. Human genome project: to sequence all 3.3 billion bases in human genome
          1. Locate genes to chromosomes
          2. Break up chromosomal DNA and attach to YAC
          3. Maps
               a. Classical – based on recombination frequencies
               b. Modern linkage – based on RFLP markers
               c. Physical – base pair distances
          4. Contigs – overlapping fragments
XII. Gene therapy
XIII. Ethics – questions about insurance, employment, etc., abound
XIV. Practical benefits
     A. Useful proteins – insulin, growth hormone
     B. Sequence analysis
     C. Identification, forensics
     D. Gene therapy
     E. Plant or animal engineering
     F. Industry – waste conversion, oil-eating microbes