DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage...
-
Upload
gloria-richardson -
Category
Documents
-
view
227 -
download
0
Transcript of DNA Recombinant. Major Steps in building a DNA Library Cloning in Plasmid Vector In bacteriophage...
![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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/1.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/2.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/3.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/4.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/5.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/6.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/7.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/8.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/9.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/10.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/11.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/12.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/13.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/14.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/15.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/16.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/17.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/18.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/19.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/20.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/21.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/22.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/23.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/24.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/25.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/26.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/27.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/28.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/29.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/30.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/31.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/32.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/33.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/34.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/35.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/36.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/37.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/38.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/39.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/40.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/41.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/42.jpg)
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.](https://reader036.fdocuments.in/reader036/viewer/2022062301/56649f255503460f94c3c1dd/html5/thumbnails/43.jpg)
Conjugation:Tripartite Conjugation:Tripartite matingmating