Library screening

Searching the genes of interest in a DNA library

1. M.SRIDEVI II.MSC (MICROBIOLOGY) 18PY15 LIBRARY SCREENING

2. DNA library is a collection of DNA fragments. It may be divided into two types. 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. Introduction

3. The genomic library is normally made by λ phage vectors, instead of plasmid vectors, for the following reasons. The entire human genome is about 3 x 10 9  bp long whereas a plasmid or λ phage vector may carry up to 20 kb fragment. This would require 1.5 x 10 5  recombinant λ phages. The maximum number to allow isolation of individual colonies is about 200 colonies per dish. Thus, at least 700 petri dishes are required to construct a human genomic library . By contrast, as many as 5 x 10 4  λ 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 the λ phage vector is that its transformation efficiency is about 1000 times higher than the plasmid vector. Genomic Library

4. f Preparation of a DNA Library Fig: Preparation of the genomic library using λ vectors. It is basically the cloning of all DNA fragments representing the entire genome

5. cDNA Library The advantage of cDNA library is that it contains only the coding region of a genome . To prepare a cDNA library, the first step is to isolate the total mRNA from the cell type of interest. Because eukaryotic mRNAs consist of a  poly-A tail , they can easily be separated. Then the enzyme reverse transcriptase is used to synthesize a DNA strand complementary to each mRNA molecule. After the single-stranded DNA molecules are converted into double-stranded DNA molecules by DNA polymerase, they are inserted into vectors and cloned.

6. Screening procedures Screening Colony and plaque hybridization Expression screening Hybrid arrest and release

7. Screening The process of identifying one particular clone containing the gene of interest from among the very large number of others in the gene library . The identification of specific clone from a DNA library can be carried out by using either ( 1)the sequence of the clone or ( 2)the structure/function of its expressed product Screening the product of a clone is applied only to expression libraries where the DNA fragment is expressed to yield proteins and the product is recognized by antibody / ligand

8. Screening libraries Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product 1. Radiolabeled probes which is complementary to a region of the interested gene 2 . Hybridize the labeled probe with DNA membrane (Southern ) or RNA (Northern) membrane.

9. Hybrid arrest and screen Individual cDNA clones or pools of clones can be used to hybridize to mRNA preparation Hybrid arrest : Translate the mRNA population directly , and the inhibition of translation of some products detected. Hybrid release translation : Purify the hybrids and the hybridized mRNAs released from them and translated, it identifies the protein encoded by the cDNA clone

10. Isolating individual clones Screening by sequence Hybridization PCR Screening by protein structure/biological function Screening libraries for specific genes

11. Screening by hybridization Very fast Applicable to a large number of clones Can identify clones that are not full length

12. Fig :Colony hybridization

13. Fig: Plaque hybridization

14. SCREENING BY PCR The PCR is widely used to isolate specific DNA sequences from genomic DNA and now It has been a useful technique for library screening . This method is first demonstrated by takumi a nd lodish in 1994 To isolate a specific clone the PCR is carried out with gene specific primers 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 are identified

15. EXPRESSION LIBRARIES SCREENING Identify the protein product of an interested gene If a DNA library is established using expression vectors, each individual clone can be expressed to yield a polypeptide. This type of screening is important where the DNA sequence of the target sequence is unknown. 1 . Protein activity 2.Western blotting using a specific antibody

16. Expression screening Antibodies can be used to screen the expression library . Procedure : ‘Plaque lift’ ( taken by placing a membrane on the dish of plaque ) Immersed in a solution of the antibody Detected by other antibodies Repeat cycles of screening to isolate pure plaques

17. Developed in 1970 when plasmid vectors are used to construct genomic libraries Immunological screening involves the use of antibodies that specifically recognize antigenic determinants on the polypeptide This technique can be applied to any protein for which an antibody is available. The molecular target for recognition is generally an Epitope . IMMUNOLOGICAL SCREENING

18. Earlier immunoscreening methods employed radio-labeled primary antibodies to detect antibody binding to the nitrocellulose sheet It is now superseded by antibody sandwiches resulting in highly amplified signals. The secondary antibody recognizes the constant region of the primary antibody & is , additionally, conjugated to an easily assayable enzyme ( e.g . horseradish peroxidase or alkaline phosphatase) which can be assayed using colorimetric change or emission of light using X-ray film .

19. procedure In this technique, the cells are grown as colonies on master plates and transferred to a solid matrix. These colonies are subjected to lysis releasing the proteins which bind to the matrix. These proteins are treated with a primary antibody which specifically binds to the protein (acts as antigen ). The unbound antibodies are removed by washing.

20. A secondary antibody is added which specifically binds to the primary antibody removing the unbound antibodies by washing. The secondary antibody carries an enzyme label (e.g., horse radish peroxidase or alkaline phosphatase) bound to it which converts colorless substrate to colored product. The colonies with positive results (i.e. colored spots) are identified and sub cultured from the master plate)

21. Fig: Schematic process of immunological screening using antibody sandwich.

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