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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