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Library screening


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Library screening

  2. 2. The identification of specific clone from a DNAlibrary can be carried out by exploiting either(1)the sequence of the clone or(2)the structure/function of its expressed product.Former type can be applied to genomic or cDNAlibraries and nucleic acid hybridization is donewith the help of probes or primers.Screening the product of a clone is applied onlyto expression libraries where the DNA fragment isexpressed to yield proteins and the product isrecognized by antibody /ligandSEQUENCE DEPENDENT SCREENINGScreening by hybridizationNucleic acid hybridization is the most commonlyused method and it is rapid
  3. 3. Grunstein &hogness (1975) developed a screening procedure to detectDNA sequences in transformed colonies by hybridization with radioactiveRNA probes.The variation of this method is devisedBy benton and davis and it is called asPlaque lift method.This method is widely used in isolatingRecombinant phage particles.Thus hybridiztion has potential toisolate any sequence if probe isavailable.A number of alternative labelingmethods are available that avoiduse of radioacticityIncorporation of digoxigenin orbiotin detected by specific antibody
  4. 4. SCREENING BY PCRThe PCR is widely used to isolate specific DNA sequences from unclonedgenomic DNA and now It has been a useful technique for libraryscreening.This method is first demonstrated by takumi . And lodish in 1994Versatile like hybridization.To isolate a specific clone the PCR is carried out with gene specificprimers that flank a unique sequence in the target.Pools of clones are maintained in multiwell plates.Each well is screened by the PCR and positive wells areidentifiedThe clone in each positive well are then identified are thendiluted into series in a secondary set of plates and screened again
  5. 5. The process is repeated until wells carryinghomogeneous clones corresponding thegene of interest have been identified.EXPRESSION LIBRARIES SCREENING.If a DNA library is established using expressionvectors, each individual clone can beexpressed to yield a polypeptide.This type of screening is important where theDNA sequence of the target sequence isunknown.
  6. 6. IMMUNOLOGICAL SCREENINGDeveloped in 1970 when plasmid vectors are used to construct genomiclibrariesImmunological screening involves the use of antibodiesthat specifically recognize antigenic determinantson the polypeptideThis technique can be applied to any protein for whichan antibody is available.The molecular target for recognition is generally anEpitope.It is a short sequence of amino acid that folds in a particular threedimensional conformation on the surface of theprotein.
  7. 7. Broome and Gilbert method was widely used.Transformed cells were plated in Petri dishes and allowed to formcolonies.The replica plating is done and colonies were lysed.A sheet of polyvinyl that had been coated with appropriate antibodywas then applied to surface allowing antigen antibody complexformationThen sheet was then removed and exposed to 125I labeled IgG and thenexposed to X-Ray film.This technique also known as sandwichtechnique and the protein must haveatleast two determinants.
  8. 8. SOUTHWESTERN AND NORTH WESTERN SCREENINGa plaque lift is carried out to transfer a print of the library ontonitrocellulose membrane .Here screening is carried out by incubating radio labeled doublestranded DNA oligonucleotide probe containing recognition sequencefor the DNA-binding protein .Combines the principles of southern and western blots. It has beenparticularly successful in the isolation of clones expressing cDNAsequences.A limitation of this technique is that since individual plaques contain onlysingle cDNA clones, transcription factors that function only in the form ofheterodimers or multimeric complex do not recognize DNA probe.The affinity of the polypeptide for specific DNA sequence must be high.To isolate RNA binding proteins single stranded RNA is used and thismethod is called as north western screening
  9. 9. Alternatively ligands can be used to identifypolypeptides that specifically bind certain molecules.It is not widely used because of low sensitivity .
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