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cunA Ǝ Cenomlc llbrarlesŦ
8vţ
SwapnllŦk
MŦScŦţ8loLechnoloavţ
2
nd
vearŦ
Culded bvţ
urŦvedhmurLhv
PCuţ
uepLŦof 8loLechnonolavţ
Cxford Colleae of Sclenceţ
8analoreŦ
ConLenLs
· lnLroducLlonŦ
· Cenomlc llbrarlesŦ
· SLoraae of aenomlc llbrarlesŦ
· AdvanLaaes Ǝ dlsadvanLaaes of aenomlc llbrarlesŦ
· AppllcaLlons of aenomlc llbrarlesŦ
· cunA llbrarvŦ
· MeLhods for svnLhesls of cunAŦ
· Screenlna proceduresŦ
÷ nuclelc acld hvbrldlsaLlonŦ
÷ 8lueŴwhlLe screenlnaŦ
· AppllcaLlons of cunA llbrarlesŦ
· ConcluslonŦ
· 8eferencesŦ
3974/:.943
· 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.
· enomic 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, / 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.
Cenomlc llbrarvŦ
· A enomic Iibrary: 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 enomic Iibraries
· /-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 10
9
bp long while
a plasmid may carry up to 20 kb fragment. This would
require 1.5 x 10
5
recombinant plasmids. When plating
.4 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 10
4
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.
$torae of Libraries.
AdvanLaaes Ǝ ulsadvanLaaes of
aenomlc llbrarlesŦ
· LnLlre aenome of an oraanlsm ls usedŦ
· Cenomlc llbrarles are noL useful whlle worklna
wlLh eukarvoLesŦ
· Screenlna someLlmes becomes dlfflculLŦ
· Poweverţ aenomlc llbrarles allow us Lo sLudv
Lhe aenome sequence of a parLlcular aeneŦ
App||cat|ons of Genom|c L|brary
Ŧ Cenomlc llbrarv consLrucLlon ls Lhe flrsL
sLep ln anv unA sequenclna pro[ecLsŦ
2Ŧ Cenomlc llbrarv helps ln ldenLlflcaLlon of
Lhe novel pharmaceuLlcallv lmporLanL aenesŦ
3Ŧ Cenomlc llbrarv helps ln ldenLlflcaLlon of
new aenes whlch were sllenL ln Lhe hosLŦ
4Ŧ lL helps us ln undersLandlna Lhe complexlLv
of aenomesŦ
cunA llbrarv
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.
9rotoco| for construct|on of
an cDNA ||braryŦ
@alllna Ǝ prlmlna meLhod
@he selfŴprlmlna meLhod
nase n method for cDNA synthes|sŦ
creen|ng procedures
· $creenin : 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.
· CIony and pIaque 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.
20-5
aster pIate
FiIter
$oIution
containin
probe
FiIter Iifted
and fIipped over
Radioactive
sinIe-stranded
DNA
Probe
DNA
ene of
interest
$inIe-stranded
DNA from ceII
FiIm
Hybridization
on fiIter
aster pIate
CoIonies
containin
ene of
interest
A speciaI fiIter paper
is pressed aainst
the master pIate,
transferrin ceIIs to
the bottom side of
the fiIter.
The fiIter is treated to break
open the ceIIs and denature
their DNA; the resuItin
sinIe-stranded DNA
moIecuIes are treated so that
they stick to the fiIter.
The fiIter is Iaid
under photoraphic
fiIm, aIIowin any
radioactive areas to
expose the fiIm
(autoradioraphy).
After the
deveIoped fiIm is
fIipped over, the
reference marks
on the fiIm and
master pIate are
aIined to Iocate
coIonies carryin
the ene of
interest.
NucIeic Acid Hybridization
· 8lue whlLe screenlnaŦ
EssentiuI feutures
PoIyIinker
SeIectubIe murker (Amp
r
)
ScreenubIe Murker (LucZ)
ßucteriuI Oriqin of repIicution (oriR)
pUC1ß - u common
cIoninq vector
Lac Z gene
ßetu-quIuctosiduse
X-quI
(coIorIess)
SuI + X(ßIue dye)
bIue coIonies
White coIonies
NO ßetu-quIuctosiduse
EcoR1
Interrupted
Lac gene
EcoR1
EcoR1
pUC18
pUC18
"Recombinunt
MoIecuIes"
LucZ- u screenubIe murker
AIIows for eusy visuuI "screeninq" of bucteriuI coIonies thut contuin
recombinunt DNA moIecuIes
ßucteriuI coIonies trunsformed with pUC1ß
bIue coIonies
(contuin non-recombinunt DNA
moIecuIes)
White coIonies
(contuin recombinunt DNA
moIecuIes)
AppllcaLlons of cunA llbrarlesŦ
· cunA llbrarles creaLes acess Lo some
oraanlsms aenomes who do noL posses a Lrue
unAŦ
· cunA llbrarles allows us Lo ellmlnaLe Lhe real
[unk ln an enLlre unAŦ
· Lxpresslons of aenes ls easvŦ
ConcluslonŦ
· 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.
· ven a single gene of interest can be easily
isolated and cloned.
· Ìt allows us to properly notify the expressions of
various genes present in the genome.
8eferencesŦ
· B.D.Singh, Biotechnology expanding horizons,
2009 edition, Kalyani publishers, 25-36.
· rnst-.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 odge , Pete und & Steve Mirchin, Gene
cloning principles & applications, Taylor & francis
group, 85-141.
IHANK y0k

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