Library screening Heterologous and homologous gene probes Differential

Library screening ü Heterologous and homologous gene probes ü Differential screening ü Expression library screening

Heterologous and homologous gene probes ü ü 1. 2. A genomic or c. DNA 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

Heterologous and homologous gene probes ü The ability of heterologous sequence to form duplexes depends on: 1. The length of matching sequence 2. Base composition 3. The number of mismatches 4. The reaction (hybridization) conditions 5. The stringency of the condition in which the probe is washed after hybridization

Type of probe ü 1. 2. 3. 4. 5. 6. 7. The probe may be a sequence which has already been cloned, such as: c. DNA PCR product Genomic fragment from a different species An incomplete part of the gene or a c. DNA to be isolated A sequence from a different gene from the same species A synthetic oligonucleotide Mixed sets of sequences derived from particular m. RNA population

Procedure ü ü ü 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

Screening a library by hybridization

Application ü ü Isolation of homologous gene sequences from the same species (use of a c. DNA probe to isolate a genomic clone, use of PCR product to isolate c. DNA or genomic clones, use of a partial gene or c. DNA 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

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

Differential screening ü Differential screening has been used to identify c. DNA clones that reflect m. RNAs present at different level in different cell types ü It is a technique that allows the analysis of differentially regulated gene transcription and the cloning of m. RNAs which are differentially transcribed between two or more samples ü 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

Methodology ü ü ü ü 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 c. DNA, which is then cloned into vector The c. DNA 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 m. RNA fraction After autoradiography, clones which show different intensities of hybridization signal with the two probes identify m. RNas 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

Expression library screening ü Rapidly screen large c. DNA libraries expressing foreign proteins using antibody probes ü It is enable us to isolate genes expressing certain proteins against which antibodies could be raised ü A number of factors can influence the successful outcome of screening c. DNA expression libraries: 1. 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 c. DNA library 2. The quality of antibody itself

Screening a library with antibodies