dna recombinant. major steps in building a dna library cloning in plasmid vector in bacteriophage...
TRANSCRIPT
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