DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage...

DNA RecombinantDNA Recombinant

Major Steps in building a Major Steps in building a DNA LibraryDNA LibraryCloning 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 EnzymeStaggered “sticky ends”

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction EnzymeRole of T4 DNA Ligase

Choosing the VectorChoosing the Vector

Depends on the size of DNA to be cloned

Is 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 MapWe 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 Kinds of plasmids in the wild: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 Characteristics of an engineered plasmid:engineered plasmid:

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

Unique restriction enzime sites for cloning

One 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 pUC19 another plasmid cloning vectorcloning vectorCharacteristics: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: Creation of a DNA Library: Partial DNA digestionPartial DNA digestion

Purpose: To produce overlapping DNA fragments

Progress of Reaction: Progress of Reaction: agarose gel electrophoresisagarose gel electrophoresisVary 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 Klenow Fragment of E. coli DNA Polymerase I Enzyme DNA Polymerase I Enzyme ActivitiesActivities 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 Screening a Genomic LibraryLibraryColony Hybridization

Colony Hybridization:Colony Hybridization:Procedur

e:

Screening a Genomic Screening a Genomic Library: II.Colony Library: II.Colony ImmunoassayImmunoassay

The probe is an antibodyThe colonies are grown so that the recombinant protein is expressed

Screening a Genomic Screening a Genomic Library: II.Colony Library: II.Colony ImmunoassayImmunoassay

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

Screening a Genomic Screening a Genomic Library:Library:III.Functional III.Functional ComplementationComplementation 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 Another Type of Library:cDNA LibraryLibrary:cDNA LibraryUsed 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: Genomic Library: Bacteriophage Bacteriophage λλ (Lambda) (Lambda)For cloning inserts of 10-20 KbPlasmid libraries hold up to 10 kb inserts

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

Cycle:Production of progeny

Lysogenic Cycle: Integration into bacterial chromosome

Genomic Library: Genomic Library: Bacteriophage Bacteriophage λλ

Genomic Library: Genomic Library: Bacteriophage 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 plasmids

It’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 Methods of Introducing Foreign DNAForeign DNATransformationElectroporationConjugation

ElectroporationElectroporation

Application of an electrical field to cells

Conjugation:Tripartite Conjugation:Tripartite matingmating