This document discusses gene and DNA libraries. It defines a gene library as a collection of DNA fragments cloned into vectors. There are two main types of libraries: genomic libraries, which contain total genomic DNA from an organism, and cDNA libraries, which contain cloned cDNA fragments representing part of an organism's transcriptome. The document outlines the steps to construct both genomic and cDNA libraries, including fragmenting DNA, ligating it into a vector, transforming the vector into host cells, and screening the library. Gene libraries have applications such as discovering novel genes, studying gene function, and producing proteins.

1. GENE LIBRARY
PRESENTED By: NIDHI SHARMA
M.S.c FINAL YEAR
16-BTM-05
GG7517

2. GENE OR DNA LIBRARY
 Collection of DNA fragments- cloned into vectors
 Researchers -identify and isolate the DNA of interest.
DNA
LIBRARY
GENOMIC
LIBRARY
cDNA
LIBRARY

3. Types of Libraries
• Collection of total genomic DNA from a single
organism.
Genomic Library
• Collection of cloned cDNA fragments constituting
some portion of the transcriptome of an organism
cDNA Library

4. CONSTRUCTION OF GENOMIC LIBRARY
Genomic DNA
DNA fragments
SUITABLE VECTOR
R.E.
R.E.
LIGATION
rDNA
SCREENING

5. C-dna library
• Cells exhibit tissue-specific gene expression.
• Not possible to clone mRNA directly.
• Cloned into a suitable vector.
• Collection of the cDNA clones - cDNA library.
mRNA cDNAREVERSE
TRANSCRIPTASE

6. STEPS.......
 1. Isolation of RNA
 2. Sepration of mRNA from total RNA
 3. Synthesis of first strand of cDNA
 4. Synthesis of 2nd strand of cDNA
 5. Cloning of double stranded cDNA
 6. Screening of cDNA library

8. • RNA – tRNA
mRNA
rRNA
• AFFINITY CHROMATOGRAPHY
with oligo-dT matrix.
mRNA binds because of
Poly-A tail.
•Wash and add ELUTION Solution
• mRNA serprated

9. mRNA
5’ 3’
REVERSE
TRANSCRIPTASE +
Primer +
dA dT dG dC
cDNA-mRNA hybrid

10. Synthesis of 2nd strand of cDNA
3 METHODS:
i. SELF-PRMING
SYNTHESIS
ii. REPLACEMENT
METHOD
iii. PRIMED METHOD

11. REPLACEMENT METHOD

12. PRIMED METHOD

13. Cloning of double stranded
cDNA
 Introduce restriction site
linkers to ends of cDNA
by blunt end ligation.
 Digestion with restriction
enzyme(R.E) to create
sticky ends.
 Mix cDNA with vector
DNA (cut with same R.E.)
in presence of DNA
ligase.
 Transform construct into
an host(E.coli or λ-Phage)
for cloning.

14. CHOICE OF VECTOR
 ACCORDING TO SIZE OF DNA to be ligated


15. SCREENING OF cDNA LIBRARY
• GRUNSTEIN AND
HOGNESS (1975) –
COLONY/PLAQUE
NUCLEIC ACID
HYBRIDISATION
TECHNIQUE

16. GENOMIC LIBRARY VS cDNA LIBRARY
GENOMIC LIBRARY
• From genomic DNA
• frequency of hits is
independent of gene
expression levels.
• may contain promoters and
introns.
• useful for genome analysis,
map-based cloning, promoter
studies, etc.
cDNA LIBRARY
• Reverse transcription of mRNA
• Dependent
• no promoters or introns.
• useful for analysis of coding
regions and gene functions

17. Application of Gene library:-
• Discovery of novel genes.
• Elucidation of gene function.
• In vitro study of gene function.
• Toobtain pure sample of a gene.
• Commercial production of proteins and other
biological molecules.
• Carcinogen identification. Etc