2012 recombinant dna methods and technology(1)
TRANSCRIPT
RECOMBINANT DNA METHODS AND TECHNOLOGY
Matthew George, Jr., Ph.D.
Recombinant DNA and Genetic EngineeringRecombinant DNA and Genetic Engineering
A recombinant DNA molecule is produced by splicing A recombinant DNA molecule is produced by splicing together DNA from different sources. It is usually done for together DNA from different sources. It is usually done for one of the following reasons:one of the following reasons:
To clone large amounts of DNA for the studies of gene To clone large amounts of DNA for the studies of gene organization; structure; sequencing; and gene regulationorganization; structure; sequencing; and gene regulation
To genetically engineer cells in an organism to confer new To genetically engineer cells in an organism to confer new desired characteristics (i.e., gene therapy, and correct desired characteristics (i.e., gene therapy, and correct genetic defects)genetic defects)
To produce the product(s) of spliced genes in bacterial cells To produce the product(s) of spliced genes in bacterial cells (i.e. insulin, human growth hormone)(i.e. insulin, human growth hormone)
Alter the genetic characteristics of fruits and vegetablesAlter the genetic characteristics of fruits and vegetables To produce organisms (i.e. Dolly, cows and mice for To produce organisms (i.e. Dolly, cows and mice for
therapeutic purposes)therapeutic purposes)
STEM CELL RESEARCH: to produce new tissues and organsSTEM CELL RESEARCH: to produce new tissues and organs
Recombinant DNA Product Effect?Recombinant DNA Product Effect?
THE UTILITY OF RECOMBINANT DNA TECHNOLOGYTHE UTILITY OF RECOMBINANT DNA TECHNOLOGY
RESTRICTION ENDONUCLEASESRESTRICTION ENDONUCLEASES
BASIC CLONING STRATEGYBASIC CLONING STRATEGY
EcoRI Restriction EndonucleaseEcoRI Restriction Endonuclease
EcoRI RecombinantEcoRI Recombinant
Vector CharacteristicsVector Characteristics
Basic components of a plasmid cloning vector Basic components of a plasmid cloning vector that can replicate within an E. coli cellthat can replicate within an E. coli cell
Molecular Cell Biology, 7th Edition, Lodish et al.
CLONING VECTORSCLONING VECTORS
Structure of pBR322 - a common cloning vector• derived from a naturally occurring plasmid•has unique restriction enzyme cleavage sites for
insertion of foreign DNA (100bp to 10kb)• has antibiotic resistance genes for selection of
transformants containing the plasmid (insertion of DNA at these sites will result in the bacteria becoming sensitive to that antibiotic)•has origin of DNA replication (ori) for propagation in E. coli
Fig. 7.7
Yeast Artificial Chromosome (YAC)
Normal ends of eukaryotic chromosomes
DNA cloning in a plasmid vector permits amplification of a DNA fragment*DNA cloning in a plasmid vector permits amplification of a DNA fragment*
See Devlin, fig. 7.9; *Molecular Cell Biology 7th Edition, Lodish et al.
Cloning a segment of DNA into a plasmid vector
• bacteria are “transformed ” with the recombinant plasmid• colonies that grow in tetracycline, but not in ampicillin are isolated
PstIHuman DNA cut with PstI
P
P
pBR322 amp R, tetR
pBR322 (human clone) tet R
P
P
ampR tetR
pBR322 DNA cut with PstIinactivating the ampR gene
tetR
tetR
combineand
ligate
AntibioticAntibioticR R SelectionSelection
cDNA Formation
A cDNA library contains representative copies of cellular mRNA sequences*A cDNA library contains representative copies of cellular mRNA sequences*
See Devlin’s figure 7.16; *Molecular Cell Biology, 7th Edition, Lodish et al.
Genomic DNA LibraryGenomic DNA Library
Lambda Phage Cloning Lambda Phage Cloning
See fig. 7.13
Northern BlottingNorthern Blotting
(Transfer of RNA from a gel to a solid matrix)(Transfer of RNA from a gel to a solid matrix)
Gives information about whether RNAs are present Gives information about whether RNAs are present in a tissue. Often used to determine whether in a tissue. Often used to determine whether particular genes are expressed. Does particular genes are expressed. Does NOTNOT give give information about whether the gene is present in information about whether the gene is present in other tissues.other tissues.
Presence of GlucokinasePresence of Glucokinase
Human Liver Human Muscle Rat Brain E. coli
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Expression VectorsExpression Vectors
Fig. 7.22
Some eukaryotic proteins can be produced in Some eukaryotic proteins can be produced in E. coli E. coli cells cells from plasmid vectors containing the from plasmid vectors containing the laclac promoter promoter
Molecular Cell Biology, 7th Edition, Lodish et al.
Detection of ExpressionDetection of Expression
PCRPCR
The CSI Effect
Sanger Dideoxy SequencingSanger Dideoxy Sequencing
Fig. 7.5
Dideoxynucleotide
Transgenic animals - the gene of interest together with appropriate regulatory elements is microinjected into a fertilized mouse egg which is then inserted into the uterus of a foster mother mouse
Microarray TechnologyMicroarray Technology
siRNA: “Gene Silencing”siRNA: “Gene Silencing”
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