Seminar oncDNA & Genomic libraries.

By,Swapnil.K

M.Sc.,Biotechnology,2nd year.

Guided by,Dr.VedhmurthyHOD,Dept.of Biotechnonolgy,Oxford College of Science,Banglore.

Contents• Introduction.• Genomic libraries.• Storage of genomic libraries.• Advantages & disadvantages of genomic libraries.• Applications of genomic libraries.• cDNA library.• Methods for synthesis of cDNA.• Screening procedures.

– Nucleic acid hybridisation.– Blue-white screening.

• Applications of cDNA libraries.• Conclusion.• References.

Introduction• Libraries: A  library is a population of

host bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the entire genome of the source organism.

• They can be– Genomic libraries.– cDNA libraries.

• Genomic DNA : For making libraries, genomic DNA, usually prepared by protease digestion and phase extraction, is fragmented randomly by physical shearing or restriction enzyme digestion to give a size range appropriate for the chosen vector. Often combination of restriction enzymes are used to partially digest the DNA.

• Vectors : Plasmids, λ phage, cosmid, BAC or yeast artificial chromosome vectors can be used to construct genomic libraries, the choice depending on the genome size. The upper size limit of these vectors is about 10,23,45,350 and 1000 kb respectively. The genomic DNA fragments are ligated to the prepared vector molecule using T4 DNA ligase.

Genomic library.• A genomic library: is a population of host

bacteria, each of which carries a DNA molecule that was inserted into a cloning vector, such that the collection of cloned DNA molecules represents the entire genome of the source organism.

• Vectors for genomic libraries• λ-phage - 9-23 kb → convenient and easy to

handle• Cosmids - 30-45kb• BAC, YAC →artificial chromosomes that handle

large inserts.• Fosmids- 40kb

• The entire human genome is about 3 x 109 bp long while a plasmid may carry up to 20 kb fragment.  This would require 1.5 x 105 recombinant plasmids. When plating E. coli colonies on a petri dish, the maximum number of individual colonies is about 200 colonies per dish.  Thus, at least 700 petri dishes are required to construct a human genomic library. 

• As many as 5 x 104  phage plagues can be screened on a typical petri dish.  This requires only 30 petri dishes to construct a human genomic library. 

• Another advantage of phage vector is that its transformation efficiency is about 1000 times higher than the plasmid vector.

Storage of Libraries.

Advantages & Disadvantages of genomic libraries.

• Entire genome of an organism is used.• Genomic libraries are not useful while working

with eukaryotes.• Screening sometimes becomes difficult.• However, genomic libraries allow us to study

the genome sequence of a particular gene.

Applications of Genomic Library

1. Genomic library construction is the first step in any DNA sequencing projects.2. Genomic library helps in identification of the novel pharmaceutically important genes.3. Genomic library helps in identification of new genes which were silent in the host.4. It helps us in understanding the complexity of genomes.

cDNA library

Advantages of cDNA libraries

There are no introns, so there is no danger of pieces of your gene being chopped onto separate clones; and the library is (hopefully) enriched for your gene, since instead of one or two copies, as in the genomic library, you have as many copies as the cell could produce mRNA's for that gene. So most molecular biologists, when searching for a new gene, start by screening a cDNA library from a tissue or organism that they suspect is actively using that gene. 

Protocol for construction of an cDNA library.

Tailing & priming method

The self-priming method

Rnase H method for cDNA synthesis.

Screening procedures• Screening : Screening to isolate one particular

clone from a gene library routinely involves using a nucleic acid probe for hybridization. The probe will bind to its complementary sequence allowing the required clone to be identified.

• Clony and plaque hybridization: A copy of the position of colonies or plaques on a petri dish is made on the surface of a membrane, which is then incubated in a solution of labeled probe. After hybridization and washing, the location of the bound label is determined. The group of colonies/plaques to which the label has bound is diluted and re-plated in subsequent rounds of screening until an individual clone is obtained.

LE 20-5

Master plate

Filter

Solutioncontainingprobe

Filter liftedand flipped over

Radioactivesingle-strandedDNA

ProbeDNA

Gene ofinterest

Single-strandedDNA from cell

Film

Hybridizationon filter

Master plate

Coloniescontaininggene ofinterest

A special filter paper is pressed against the master plate, transferring cells to the bottom side of the filter.

The filter is treated to break open the cells and denature their DNA; the resulting single-stranded DNA molecules are treated so that they stick to the filter.

The filter is laid under photographic film, allowing any radioactive areas to expose the film (autoradiography).

After the developed film is flipped over, the reference marks on the film and master plate are aligned to locate colonies carrying the gene of interest.

Nucleic Acid Hybridization

• Blue white screening.

Essential featuresPolylinkerSelectable marker (Ampr)Screenable Marker (LacZ)Bacterial Origin of replication (oriR)

pUC18 - a common cloning vector

Lac Z gene

Beta-galactosidaseX-gal(colorless)

Gal + X(Blue dye)blue colonies White colonies

NO Beta-galactosidase

EcoR1

InterruptedLac gene

EcoR1 EcoR1

pUC18 pUC18

“Recombinant Molecules”

LacZ- a screenable marker

Allows for easy visual “screening” of bacterial colonies that contain recombinant DNA molecules

Bacterial colonies transformed with pUC18

blue colonies(contain non-recombinant DNA molecules)

White colonies(contain recombinant DNA molecules)

Applications of cDNA libraries.

• cDNA libraries creates acess to some organisms genomes who do not posses a true DNA.

• cDNA libraries allows us to eliminate the real junk in an entire DNA.

• Expressions of genes is easy.

Conclusion.

• The genomic library contains DNA fragments representing the entire genome of an organism.

• The cDNA library contains only complementary DNA molecules synthesized from mRNA molecules in a cell.

• They are important as they provide a gateway for cloning.

• Even a single gene of interest can be easily isolated and cloned.

• It allows us to properly notify the expressions of various genes present in the genome.

References.

• B.D.Singh, Biotechnology expanding horizons, 2009 edition, Kalyani publishers, 25-36.

• Ernst-L.Winnacker, From genes to genomes, 2003 edition, Panima publishing house, 32-46.

• Jeremy.W.Dale & Malcolm von Schantz, From genes to genomes concept & applications, 2002 edition, British library publications, 99-116.

• Julia Lodge , Pete Lund & Steve Mirchin, Gene cloning principles & applications, Taylor & francis group, 85-141.

THANK YOU