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RECOMBINANT DNA • DNA THAT CONTAINS DNA SEGMENTS OR GENES FROM DIFFERENT SOURCES. • DNA TRANSFERRED FROM ONE PART OF A DNA MOLECULE TO ANOTHER, FROM ONE CHROMOSOME TO ANOTHER CHROMOSOME, OR FROM ONE ORGANISM TO ANOTHER ALL CONSTITUTE RECOMBINANT DNA • RECOMBINANT DNA TECHNOLOGY THE TRANSFER OF DNA SEGMENTS ARTIFICIALLY DNA CLONING • METHODS FOR PREPARING WELLDEFINED, GENE-SIZED PIECES OF DNA IN MULTIPLE IDENTICAL COPIES • BACTERIA AND THEIR PLASMIDS HAVE BEEN USED A LOT FOR CLONING STUDIES BACTERIAL PLASMIDS AND CLONING RESTRICTION ENZYMES • MAJOR TOOLS IN RECOMBINANT DNA TECHNOLOGY • THESE ENZYMES OCCUR NATURALLY IN BACTERIA (THEY PROTECT BACTERIUM FROM INTRUDING DNA FROM OTHER ORGANISMS) • RESTRICTION ENZYMES ARE VERY SPECIFIC, CUTTING DNA AT SPECIFIC RECOGNITION SEQUENCES OF NUCLEOTIDES. RESTRICTION ENZYMES AND DNA LIGASE STICKY ENDS • THE CUT ACROSS A DOUBLE-STRANDED DNA IS USUALLY STAGGERED, PRODUCING FRAGMENTS THAT HAVE ONE STRAND OF THE DNA EXTENDING BEYOND THE COMPLEMENTARY STRAND. THE UNPAIRED EXTENSION IS CALLED A STICKY END RECOMBINANT DNA VECTORS • MOST DNA TECHNOLOGY PROCEDURES USE CARRIERS OR VECTORS FOR MOVING DNA FROM TEST TUBES BACK INTO CELLS • TWO MOST OFTEN USED TYPES OF VECTORS ARE BACTERIAL PLASMIDS AND VIRUSES PLASMIDS AS VECTORS • FIRST, THE PLASMID IS TREATED WITH THE SAME RESTRICTION ENZYME AS WAS USED TO CREATE THE DNA FRAGMENT • THE RESTRICTION ENZYME WILL CUT THE PLASMID AT THE SAME RECOGNITION SEQUENCES, PRODUCING THE SAME STICKY ENDS CARRIED BY THE FRAGMENTS • MIXING THE FRAGMENTS WITH THE CUT PLASMIDS ALLOWS BASE-PAIRING AT THE STICKY ENDS. • APPLICATION OF DNA LIGASE STABILIZES THE ATTACHMENT. • THE RECOMBINANT PLASMID IS THEN INTRODUCED INTO A BACTERIUM BY TRANSFORMATION CLONING A HUMAN GENE IN A PLASMID VIDEO:: CLONING USING PLASMID AS A VECTOR QuickTime™ and a Cine pak decomp ress or are nee ded to s ee this picture. E. COLI & INSULIN • THE HUMAN GENE FOR INSULIN HAS BEEN INSERTED INTO E. COLI. • THE TRANSFORMED E. COLI PRODUCE INSULIN WHICH IS ISOLATED AND USED TO TREAT DIABETES POLYMERASE CHAIN REACTION (PCR) • PCR IS A TECHNIQUE THAT ALLOWS ANY PIECE OF DNA TO BE QUICKLY COPIED IN VITRO • PCR USES SYNTHETIC PRIMERS THAT INITIATE REPLICATION AT SPECIFIC NUCLEOTIDE SEQUENCES • PCR IS BEING USED BY THE HUMAN GENOME PROJECT TO PRODUCE LINKAGE MAPS WITHOUT THE NEED FOR LARGE FAMILY PEDIGREE ANALYSIS GEL ELECTROPHORESIS • IN THIS PROCESS, DNA FRAGMENTS OF DIFFERENT LENGTHS ARE SEPARATED AS THEY DIFFUSE THROUGH A GELATINOUS MATERIAL UNDER THE INFLUENCE OF AN ELECTRIC FIELD • SINCE DNA IS NEGATIVELY CHARGED ( BECAUSE OF THE PHOSPHATE GROUPS), IT MOVES TOWARD THE POSITIVE ELECTRODE **SHORTER FRAGMENTS MIGRATE FURTHER THROUGH THE GELL THAN LONGER, HEAVIER FRAGMENTS. **GEL ELECTROPHORESIS IS OFTEN USED TO COMPARE DNA FRAGMENTS OF CLOSELY RELATED SPECIES IN AN EFFORT TO DETERMINE EVOLUTIONARY RELATIONSHIPS ***METHODS BOX PAGE 374 USING RESTRICTION FRAGMENT PATTERNS TO DISTINGUISH DNA FROM DIFFERENT ALLELES RFLP’S • WHEN RESTRICTION FRAGMENTS BETWEEN INDIVIDUALS OF THE SAME SPECIES ARE COMPARED, THE FRAGMENTS DIFFER IN LENGTH BECAUSE OF POLYMORPHISMS, WHICH ARE SLIGHT DIFFERENCES IN DNA SEQUENCES • THESE FRAGMENTS ARE CALLED RESTRICTION FRAGMENT LENGTH POLYMORPHISMS, OR RFLP’S • IN DNA FINGERPRINTING, PFLP’S PRODUCED FROM DNA LEFT AT A CRIME SCENE ARE COMPARED TO RFLP’S FROM THE DNA OF SUSPECTS ***METHODS BOX P. 375 RFLP MARKERS CLOSE TO A GENE COMPLEMENTARY DNA (cDNA) • WHEN FOREIGN GENES ARE INSERTED INTO THE GENOME OF A BACTERIUM WITH RECOMBINANT DNA TECHNOLOGY, INTRONS OFTEN PREVENT THEIR TRANSCRIPTION • TO AVOID THIS PROBLEM, THE DNA FRAGMENT BEARING THE REQUIRED GENE IS OBTAINED DIRECTLY FROM THE mRNA THAT CODES FOR THE DESIRED POLYPEPTIDE • REVERSE TRANSCIPTASE (OBTAINED FROM RETROVIRUSES) IS USED TO MAKE A DNA MOLECULE DIRECTLY FROM THE mRNA. • DNA OBTAINED IN THIS MANNER IS CALLED COMPLEMENTARY DNA-cDNA. DNA MICROASSAY FOR GENE EXPRESSION ONE TYPE OF GENE THERAPY-BONE MARROW TISSUE USING THE Ti PLASMID AS A VECTOR FOR GENETIC ENGINEERING IN PLANTS