Library screening

1. Library screening Heterologous and homologous gene probes Differential screening Expression library screening

2. Heterologous and homologous gene probes • A genomic or cDNA library whether in plasmid or in a lambda vector contains a large number of different recombinants • There are number of methods for screening libraries for specific sequences • The basis of the procedure is the ability of two stranded DNA molecules of complementary or nearly complementary sequence to form a duplex • Probe (a labeled single stranded DNA molecule) • Homologous: identical sequences • Heterologous: not identical sequences

3. Heterologous and homologous gene probes • The ability of heterologous sequence to form duplexes depends on: • The length of matching sequence • Base composition • The number of mismatches • The reaction (hybridization) conditions • The stringency of the condition in which the probe is washed after hybridization

4. Type of probe • The probe may be a sequence which has already been cloned, such as: • cDNA • PCR product • Genomic fragment from a different species • An incomplete part of the gene or a cDNA to be isolated • A sequence from a different gene from the same species • A synthetic oligonucleotide • Mixed sets of sequences derived from particular mRNA population

5. Procedure • Plates are prepared with either bacterial colonies containing a plasmid library or with plaques from a phage library growing on bacteria • Colonies are replicated on nitrocellulose or other hybridization filter and grown further, whereas plaques are lifted onto filter and processes immediately • The filter are processed to lyse the bacterial cells or to break open (disrupt) the phage protein coat and denature and fix the DNA in situ • There are then probed with a labeled DNA fragment which will hybridize to the sequence of interest • The position of hybridization signal is determined by autoradiography and used to identify the colony or plaque containing the sequence • The colony or plaque is picked from the master plate and replate at a density to give individual colonies or plaques and screened again

6. Screening a library by hybridization

7. Application • Isolation of homologous gene sequences from the same species (use of a cDNA probe to isolate a genomic clone, use of PCR product to isolate cDNA or genomic clones, use of a partial gene or cDNA sequence to isolate a full-length sequence • Identification of closely related gene in a gene family • Isolation of related genes from other species • Isolation of genes encoding proteins which have been completely or partially sequence. The protein sequence is back-translated to give a DNA sequence and it is used to design an oligo-nucleotide probe

8. Differential screening • The isolation of NA sequences from a library by means of differential hybridization with complex NA probes • The principle is based on differences in the concentration of NA species between two or more samples • mRNas are the NA sequences studied • Differential screening aims at isolating differentially transcribed mRNA • Differential screening represents a means to isolate NA sequences on the basis of a common regulatory mechanism rather than their identities or function

9. Differential screening • Differential screening has been used to identify cDNA clones that reflect mRNAs present at different level in different cell types • It is a technique that allows the analysis of differentially regulated gene transcription and the cloning of mRNAs which are differentially transcribed between two or more samples • Samples may be different cell types, tissues, organs, the same cells which have been subjected to different treatment, cells which have a different metabolic or physiological status

10. Methodology • Cellular RNA is extracted from samples • Poly(A) RNAs are then purified from the total RNAs and poly(A) RNA fraction obtained from one sample is used as a template for the synthesis of the corresponding cDNA, which is then cloned into vector • The cDNA library is then plated at a relatively low density to facilitate subsequent identification of individual clones by colonies or plaques hybridization • Two replica filters are taken from the master plate and hybridized independently to labeled probes obtained by reverse transcription of the mRNA fraction • After autoradiography, clones which show different intensities of hybridization signal with the two probes identify mRNas for differentially transcribed genes • The clones are then selected from master plate and usually subjected to a second round of differential hybridizaton to confirm the results obtained in the first round and eliminated the artefacts • The selected clones are characterized

11. Expression library screening • Rapidly screen large cDNA libraries expressing foreign proteins using antibody probes • It is enable us to isolate genes expressing certain proteins against which antibodies could be raised • A number of factors can influence the successful outcome of screening cDNA expression libraries: • The protein one is looking for must be expressed in the tissue which is used to extract the RNA needed for the construction of the cDNA library • The quality of antibody itself

12. Screening a library with antibodies