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 WELL- DEFINED, 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

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