C-DNA

cDNA LIBRARY& its CONSTRUCTION: 

cDNA LIBRARY& it s CONSTRUCTION Bhawana pancholi M.Sc. Biotechnology Jayoti Vidyapeeth Women’s University -Jaipur

CONTENTS: 

CONTENTS DNA library cDNAs Key points of cDNA synthesis Methods of cDNA synthesis Library construction Applications References

What is a DNA Library? : 

What is a DNA L ibrary? Genomic Library: Bank of DNA that represents the genome of specific cell/organism/species cDNA Library: Bank of cDNA representing mRNA of a specific cell. Main use - to isolate/clone DNA sequences/genes

cDNAs : 

cDNAs cDNA is the abbreviation for complementary DNA. • A cDNA library is a set of clones representing as many as possible of the mRNAs in a given cell type at a given time – Such a library can contain tens of thousands of different clones

Cont…: 

Cont… Prepared by reverse transcription of mRNA Eukaryotic mRNAs--lack introns, cDNAs of these RNAs indicate how genes are actually expressed Individual mRNA abundance varies widely: to isolate low abundance mRNAs by cDNA cloning, need to make libraries

PowerPoint Presentation: 

Key points of cDNA synthesis Isolation of mRNA Reverse transcription Synthesis of second strand of cDNA

PowerPoint Presentation: 

Reverse transcription Reverse transcriptase enzyme is used mRNA has a poly A tail mRNA is treated with oligo dT primer

PowerPoint Presentation: 

Methods of cDNA synthesis Two method s Replacement synthesis method Self priming method

PowerPoint Presentation: 

The 3 general steps used to clone cDNA Make ds cDNA of an organism 2 Cut the organismal cDNA and the vector with restriction enzymes making recombinant cDNA 3 Introduce the recombinant cDNA into a host

PowerPoint Presentation: 

Library construction 1 ) Synthesize first strand cDNA 2) Second strand cDNA 3) Methylate cDNA 4) Attach linkers or adaptors for cloning 5) Fractionate cDNA by size (select 2-8 kb) 6) Ligate cDNA into suitable vector

PowerPoint Presentation: 

Different types of cloning vectors Plasmids: Circular DNA molecules which replicate separately from the host chromosome. Plasmids used for genomic and cDNA cloning. Bacterial host. Insert size range < 15kb. 2. Bacteriophage-based Cosmids: Linear DNA molecules used for genomic and cDNA cloning. Bacterial host. Insert size range < 20kb.

PowerPoint Presentation: 

cDNA

PowerPoint Presentation: 

Applications of cDNA library Discovery of novel genes In vitro study of gene function Study of alternative splicing In carcinogen identification

PowerPoint Presentation: 

Cont … To obtain pure sample of a gene To get high yields of recombinant cDNA Elucidation of gene function Commercial production of proteins & other biological molecules