BIOL 212 General Genetics Spring 2007Lecture 23: Recombinant DNA RevisitedReading: Chap. 10 pp. 358-368Lecture Outline:1. Genetic Engineering Tools 2. Vectors3. cDNA4. Screening Lecture:1. Genetic Engineering ToolsGenetic Engineering, (aka cloning or recombinant DNA): fusion of DNAs from two (or more) organisms.Goals of G.E.:- Purify rare gene OR- Prepare rare proteinG.E. relevance to humans:- Map human genome- Isolate disease geneso Find out the proteins they encode- Prepare medicines:o Gene therapy: DNAo Drug/hormone therapies: proteins- Food, food products or additives: GMO organisms, peptides, proteins Key tools of genetic engineering:- Host organism: Especially bacteria, yeast- Vector: plasmid, virus, or artificial chromosome that carries the foreign DNA- Foreign DNA: Any organism- Restriction enzymes: “Cut DNAs”; site specific DNA endonucleases- DNA ligase enzyme: “Paste DNAs”; seal sugar-phosphate backbone of DNA2. Vectors: carry foreign DNA, allow foreign DNA to be replicated and often also transcribed and translated in recipient cells.1Types of vectors:- Plasmids- Viruses- Large capacity vectors: Cosmids, BACs, YACsPlasmid vectors: Mostly derived from E. coli plasmidsUseful features of plasmid vectorsOri site (plasmid origin of replication) - Allows plasmid to replicate in bacterial host, separate from bacterial chromosome- Derived from COLE1 plasmid of E. coliAmpicillin resistance geneMultiple cloning site (MCS), also called a polylinker- Cleavage sites for many different restriction enzymesUseful genetic marker such as lac Z gene or another antibiotic resistance gene- Enables rapid selection of cells containing recombinant DNA vs. cells with vectoralone.Fig. 10.9 pBluescript plasmid (commercial vector)Steps used in cloning:1. Cut foreign DNA with restriction enzyme2. Cut vector DNA with same enzyme or one that produces compatible ends3. Mix together, add DNA ligase to create recombinant DNA4. Use transformation to introduce gene into bacteria5. Genetic selection to distinguish recombinants from non-recombinants6. Identify desired recombinant DNA (screening see below)pBluescript: uses Blue/White color screenRecombinant DNA: Foreign DNA is inserted into the lac Z+ gene  becomes lac Z-Screen for beta-galactosidase by adding X-gal to the growth medium. Blue color indicates X-gal is present. (Fig. 10.10)Recombinant DNA present: colony is whiteNon-recombinant plasmid vector: colony is blueVirus vectors:Most common are modified E. coli virusesExample:Lambda: replace middle region genes (lysogenic region) with foreign DNA2Large capacity cloning vectors:- Needed for cloning large genes of higher organisms (for DNA regions 50 kb or larger) and for genome projects.- Can be modified forms of E. coli plasmids (cosmids), viruses (PACs), or F factors(BACs) or can be artificial yeast chromosomes (YACs)3. cDNA: Complementary DNA, a DNA copy of an mRNAMost genes from higher eukaryotes are very large.Length of mRNA much smaller than gene.Much easier to study a DNA copy of an mRNA (called a complementary DNA)Preparation of a cDNA: Fig. 10.8a. purify mRNA by affinity chromatography (most eukaryotic mRNAs have poly A tail)b. use reverse transcriptase, primer, and dNTPs to synthesize a strand of cDNAc. remove the mRNA (treat with alkali or RNase)d. use DNA polymerase I to synthesize the second strand of cDNAOR use Taq polymerase, primers and PCR to make many copies of the cDNA by PCR (this is RT-PCR or reverse transcriptase PCR)cDNA can be cloned and sequenced (may be called EST, for expressed sequence tag)4. Screening: Identify the recombinant DNA of interest- Especially important when you start with many possible recombinants:- A library consists of many bacteria, each with a different foreign DNA.Genomic library: All the DNA of an organismcDNA library: Copies of all the mRNA from a tissue or cell typeHybridization is important technique used to isolate a particular DNA of interestsome examples:- Use cDNA probe to mouse globin to detect the mouse gene- Use a mouse gene probe to detect the human geneRapid screening, indirect:Use PCR test or restriction digests to produce band of the size expected if the correct DNA was cloned.Final confirmation of recombinant DNA: Use DNA sequencing and bioinformatics