Download recombinant dna technology

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