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Page 1: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

DNA RecombinantDNA Recombinant

Page 2: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Major Steps in building a Major Steps in building a DNA LibraryDNA LibraryCloning in Plasmid VectorIn bacteriophage vectorScreening the library DNA probe Antibody probe

Page 3: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening 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

Page 4: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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”

Page 5: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Restriction Enzyme Mechanisms: Preparation of DNAs to be joined:

(b) Blunt End

Page 6: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Ligation of DNA cut with a Ligation of DNA cut with a Restriction EnzymeRestriction EnzymeStaggered “sticky ends”

Page 7: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 8: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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?

Page 9: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Restriction Enzyme MapRestriction Enzyme MapAllows ordering of DNA

fragments

Page 10: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Restriction Enzyme MapRestriction Enzyme MapWe can then build maps of linear

and circular molecules

Page 11: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 12: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Selectable Markers:Selectable Markers:

Page 13: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 14: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 15: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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.

Page 16: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 17: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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)

Page 18: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 19: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Purpose: To produce overlapping DNA fragments

Page 20: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Progress of Reaction: Progress of Reaction: agarose gel electrophoresisagarose gel electrophoresisVary time of digestion or amount

of enzyme units

Page 21: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 22: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 23: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 24: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 25: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Screening a Genomic Screening a Genomic LibraryLibraryColony Hybridization

Page 26: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Colony Hybridization:Colony Hybridization:Procedur

e:

Page 27: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 28: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 29: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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.

Page 30: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 31: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

from mRNA of specific cells

Page 32: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

cDNA Library:cDNA Library:Third

Step:Selecting and Cloning Full length cDNA molecules

Page 33: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 34: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Cycle:Production of progeny

Lysogenic Cycle: Integration into bacterial chromosome

Page 35: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Genomic Library: Genomic Library: Bacteriophage Bacteriophage λλ

Page 36: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Genomic Library: Genomic Library: Bacteriophage Bacteriophage λλ

Page 37: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Cosmid libraryCosmid library

Allows cloning of 45 kbDNA fragments

Page 38: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

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

Page 39: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

BACs: Bacterial Artificial BACs: Bacterial Artificial ChromosomesChromosomes

Page 40: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

YACs:Yeast Artificial YACs:Yeast Artificial ChromosomesChromosomes

Page 41: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Methods of Introducing Methods of Introducing Foreign DNAForeign DNATransformationElectroporationConjugation

Page 42: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

ElectroporationElectroporation

Application of an electrical field to cells

Page 43: DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage vector Screening the library DNA probe Antibody probe

Conjugation:Tripartite Conjugation:Tripartite matingmating