Gene Libraries- Genomic library, construction of genomic library, screening. applications. cDNA library, construction of cDNA library, screening, applications. by MSC MICROBIOLOGY STUDENT- Falguni Agrawal, Barkatullah University, Bhopal

1. ~ By Falguni Agrawal

2. GENES
 (1865) Gregor Mendel – Each heritable property of
an organism is controlled by a “Factor”(later called as
“gene”)
 (1903) Walter Sutton – Proposed that these factors
reside on chromosomes.
 (1909) Wilhelm Johannsen – Coined the term “Gene”
to describe the functional unit of heredity.
 (1910) T. H. Morgan – Developed techniques for gene
mapping
 (1944) Avery, Macleod, McCarty and (1952) Hershey
& chase – proved DNA as a genetic material Each set of chromosomes contain
50,000 to 100,000 genes carried in 3
billion nucleotide pairs of DNA.

3. GENE LIBRARY
 Based on from
where the segments
of DNA are derived,
gene library can be
of 2 types:
a) Genomic library
b) cDNA library
 The first ever Gene
library was
constructed by
Fredrick Sanger in
1977 of the
bacteriophage, phi
X 174 .
GENE LIBRARY
Collection of bacterial
cells
Each bacterial cell
Vector
Genomic
DNA
cDNA

4. GENOMIC LIBRARY
 Genome - an organism’s complete set of DNA, including
all of its genes.
 Genomic library - collection of cloned, restriction enzyme
digested DNA fragments containing at least one copy of
every DNA sequence present in genome of an organism.
 Factors to be considered and requirements needed
before constructing a genomic library :
a) Organism of interest
b) Restriction Enzymes
c) Appropriate vector needs to be chosen based on the
organism’s genome size.
d) Ligase enzyme
e) The necessary number of recombinant DNA
molecules has to be calculated.

5. CONSTRUCTION OF GENOMIC
LIBRARY
 The basic steps are as follows :
1. Isolation of target DNA
2. Fragmentation of DNA
3. Vector preparation
4. Ligation and introduction into host
5. Storage of genomic library
6. Screening of genomic library

6. 1. Isolation of genomic DNA

7. 2. Fragmentation of DNA
a) Mechanical shearing – using sonication or nebulizer
b) Nuclease treatment – using Restriction Endonucleases
 Fragments treated with phosphatase – to remove
terminal phosphate
Sau3A
- Consists four-base-pair
recognition site
- partial digestion

8. 3. Vector preparation
4. Ligation and
introduction into
host
5. Storage of
genomic library
6. Screening of
genomic library
a) Screening by
colony hybridization

9. B) Screening by
plaque
hybridization

10. Applications of Genomic Library
1. Genomic library construction is the first step in any DNA sequencing
projects
2. Genomic library helps in identification of novel pharmaceutically
important genes.
3. Helps in identification of new genes which were silent in the host.
4. It helps in understanding of complexity of genomes.
5. Can serve as a source of genomic sequence for generation of
transgenic animals
6. To study genetic mutations in cancer tissues

11. Hierarchical sequencing
-This sequencing
was used for the
Human Genome
Project

12. Genome wide association study(GWA
Study)
 A genome-wide association study (GWA study, or GWAS), also known as whole
genome association study (WGA study, or WGAS).
 An observational study of a genome-wide set of genetic variants in different individuals.
 To see if any variant is associated with a trait. GWASs typically focus on associations
between single-nucleotide polymorphisms (SNPs) and traits like major human diseases.
 The first successful GWAS published in 2002 studied myocardial infarction.
 This study design was then implemented in the landmark GWA 2005 study investigating
patients with age-related macular degeneration, and found two SNPs with significantly
altered allele frequency compared to healthy controls.
 The international HapMap Project is also based on this data.

13. cDNA Library
 cDNA Library - collection of cloned, restriction
enzyme digested cDNA fragments where the
cDNA represents all of the mRNA present at a
particular stage in an organism or else all the
genes that are expressed in the cells at different
stages of its development.
 Steps in the construction of cDNA Library :
1. Isolation of mRNA
2. Preparation of complementary DNA fragments
3. cloning in suitable vector system
4. Transformation in suitable host .
5. Screening of cDNA

14. 1. Isolation of mRNA
i) Lysis of cell using lysis
buffer containing detergent
ii) Centrifuge the mixture and
collect the supernatant
iii) Pass the mixture for column
chromatography

15. 2. Preparation of cDNA Fragments
 Multiple approaches have been
developed to prepare
complementary DNA (cDNA) from
isolated mRNA.
 In all approaches the 3 steps are
performed :
1. First strand synthesis with a
reverse transcriptase.
2. Removal of RNA template
3. Second strand synthesis

16. 3. cloning in suitable vector
system
 The cDNA is ligated into
the suitable vector to
generate clone
4. Transformation in suitable
host .
 Post ligation, clones are
transformed in a suitable
host to get colonies.

17. 5. Screening
of cDNA
A) Screening of
specific cDNA
plasmid in
cDNA library
using an
antibody probe
B ) Screening
using
radiolabelled
probe

18. C) Screening using colony and plaque
hybridization

19. Applications of cDNA Library
1. Discovery of novel genes.
2. Cloning of full-length cDNA molecules for in vitro study of gene function.
3. Isolating cDNAs allows to use the cDNA to develop expression vectors so
proteins of interest can be produced in high quantities, greatly simplifying the
task of protein purification.
(The human insulin gene or the corresponding cDNA has been inserted into the
early region of a simian virus 40 vector in which all SV40 splice junctions were
deleted while the early promoter and polyadenylation regions remained intact.
The expression of insulin-coding sequences was tested in permissive monkey
COS cells.)
4. A great deal of information can be deduced about the possible structure and/or
function of the protein encoded by a known cDNA

20. 5. Knowing the cDNA sequence of a protein will frequently facilitate the
development of antibodies and monoclonal antibodies.
6. cDNA sequence can be used as a probe to screen genomic libraries
and isolate the gene encoding a particular cDNA

21. REFERENCES
1. Gene Cloning and DNA analysis – T.A. Brown (6th Edition )
2. Molecular Biology – by H.D. Kumar ( 2nd revised Edition )
3. https://www.sciencedirect.com/topics/medicine-and-
dentistry/gene-library
4. https://ghr.nlm.nih.gov/primer/hgp/genome
5. Fundamentals of Genetics – Peter J. Russell (2nd Edition)
6. https://nptel.ac.in/content/storage2/courses/102103045/do
wnload/mod3.pdf
7. https://en.wikipedia.org/wiki/Genomic_library
8. https://en.wikipedia.org/wiki/Genome-
wide_association_study