Recombinant DNA Technology Prof. Elena A. Carrasquillo Chapter 4 Molecular Biotechnology Lecture 4

Recombinant DNA Recombinant DNA TechnologyTechnology

Prof. Elena A. CarrasquilloChapter 4

Molecular BiotechnologyLecture 4

Major Steps in building a DNA Major Steps in building a DNA LibraryLibrary

Cloning in Plasmid VectorIn bacteriophage vectorScreening the library DNA probe Antibody probe

Kinds of LibrariesKinds of Libraries

Genomic Library: Stores a representation of the genome (at least one copy of a gene present)

plasmid, bacteriophage, phagemid, cosmid vectorscDNA Library: Stores a representation of

the mRNAs expressed at a certain time or stage by a microorganism or organism

plasmid, bacteriophage, phagemid, cosmid vectors

How is a Library Built:How is a Library Built:Restriction Enzyme Mechanisms: Preparation of DNAs to be joined (a)Staggered cut: leaves “sticky ends”

How is a Library Built:How is a Library Built:

Restriction Enzyme Mechanisms: Preparation of DNAs to be joined:

(b) Blunt End

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme

Staggered “sticky ends”

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction Enzyme

Role of T4 DNA Ligase

Choosing the VectorChoosing the Vector

Depends on the size of DNA to be clonedIs the protein encoded by the DNA going

to be expressed in a prokariotic or eukaryotic cell?

Restriction Enzyme MapRestriction Enzyme MapAllows ordering of DNA fragments

Restriction Enzyme MapRestriction Enzyme Map

We can then build maps of linear and circular molecules

Plasmid: it’s a circular DNA Plasmid: it’s a circular DNA molecule containing:molecule containing:

a multiple cloning site or MCS an antibiotic resistance gene an origin of replication pBR322 is the basis of most engineered plasmids

Selectable Markers:Selectable Markers:

Kinds of plasmids in the wild:Kinds of plasmids in the wild:F plasmids-transfer information from cell

to cellR plasmids-confer antibiotic resistanceDegradative plasmids-utilization of

unusual metabolitesCryptic plasmids-No apparent function

Other characteristics:Other characteristics:Size range form less than 1 kb to more

than 500 kbOrigin of replication-allows plasmid to

replicate in the bacteriaLow-copy number: 1-4 per cellHigh copy number: 10-100 per cellIncompatibility groups: different kinds

cannot be inside the same cell

Characteristics of an engineered Characteristics of an engineered plasmid:plasmid:

Small size (<15Kb) for optimal efficiency of transformation in bacteria

Unique restriction enzime sites for cloningOne or more selectable genetic markers

to allow for differentiation of the plasmids carrying the cloned DNA vs the religated ones.

Plasmid: a cloning vector or Plasmid: a cloning vector or vehiclevehicle

pUC19 another plasmid cloning pUC19 another plasmid cloning vectorvectorCharacteristics:Interruption of b-lactamase gene gives

rise to white colonies (cloned DNA) vs blue ones (empty)

MCS: multiple cloning site of MCS: multiple cloning site of pUC19:pUC19:

-Produced by site-directed mutagenesis to alter the DNA sequence but not the protein sequence of b-lactamase-This produced new restriction enzyme sites

Creation of a DNA Library: Partial Creation of a DNA Library: Partial DNA digestionDNA digestion

Purpose: To produce overlapping DNA fragments

Progress of Reaction: agarose gel Progress of Reaction: agarose gel electrophoresiselectrophoresis

Vary time of digestion or amount of enzyme units

Screening a library:I. Screening a library:I. Colony Colony hybridization or Southern Blothybridization or Southern Blot

Preparation of DNA ProbePreparation of DNA ProbeMethod 1Random primersEnzyme: Klenow Fragment + dNTPsNon-radioactive: biotin or chemiluminescent labeled dNTPs-Radioactive:32P

Preparation of DNA ProbePreparation of DNA ProbeMethod 2: 5’-end labelingMethod 3: 3’end labeling

Klenow Fragment of E. coli DNA Klenow Fragment of E. coli DNA Polymerase I Enzyme ActivitiesPolymerase I Enzyme Activities

The polymerase (red) adds deoxyribonucleotides to the 3’- hydroxyl groups of the growing chains -The 5’ exonuclease (blue) removes succesive nucleotides from the 5’ phosphate ends -The 3’ exonuclease (yellow) removes succesive nucleotides from the3’ hydroxyl ends

Screening a Genomic LibraryScreening a Genomic Library

Colony Hybridization

Colony Hybridization:Colony Hybridization:Procedure:

Screening a Genomic Library: Screening a Genomic Library: II.Colony ImmunoassayII.Colony ImmunoassayThe probe is an antibodyThe colonies are grown so that the recombinant protein is expressed

Screening a Genomic Library: Screening a Genomic Library: II.Colony ImmunoassayII.Colony Immunoassay

If chemiluminescense was used, light will be emitted and captured in an X-ray film

Screening a Genomic Library:Screening a Genomic Library:III.Functional ComplementationIII.Functional Complementation

Defective host cells (A-) are transformed with a genomic library derived from cells that are normal with respect to that function (A+) and grown in minimal media.

Those cells harboring a plasmid that corrects the defect will grow in minimal media.

Another Type of Library:cDNA Another Type of Library:cDNA LibraryLibraryUsed to obtain

functional eukaryotic coding regions.

E. coli does not process introns.

First step: Isolate poly A+ mRNA with oligo (dT) cellulose

cDNA Library:cDNA Library:Second Step:Synthesis of cDNA from

mRNA of specific cells

cDNA Library:cDNA Library:Third

Step:Selecting and Cloning Full length cDNA molecules

Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ (Lambda)(Lambda)For cloning inserts of 10-20 KbPlasmid libraries hold up to 10 kb inserts

Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ (Lambda)Life Cycle(Lambda)Life Cycle

Lytic Cycle:Production of progeny

Lysogenic Cycle: Integration into bacterial chromosome

Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ

Genomic Library: Bacteriophage Genomic Library: Bacteriophage λλ

Cosmid libraryCosmid library

Allows cloning of 45 kbDNA fragments

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomesBased on P1 bacteriophage, the F plasmid

and the lacZ region of pUC plasmidsIt’s a low copy number plasmidCarries 50-300kb fragments

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes

YACs:Yeast Artificial YACs:Yeast Artificial ChromosomesChromosomes

Methods of Introducing Foreign Methods of Introducing Foreign DNADNATransformationElectroporationConjugation

ElectroporationElectroporation

Application of an electrical field to cells

Conjugation:Tripartite matingConjugation:Tripartite mating