Genomic DNA And Complementary DNA Libraries construction.

1. YBT201-RECOMBINANT DNA
TECHNOLOGY
TOPIC: GENOMIC AND CDNA
LIBRARIES CONSTRUCTION
PRESENTED BY
NAME:P. KARTHIKA
REGISTER NO:123011356010
CLASS :M.SC BIOTECHNOLOGY (1st year)
UNIVERSITY NAME: PERIYAR MANIAMMAI UNIVERSITY

2. CONTENTS
• INTRODUCTION
• DEFINITIONS
• cDNA LIBRARIES CONSTRUCTION
• GENOMIC DNA CONSTRUCTION
• CONCLUSION
• REFERENCES
SUBMITTED BY
NAME: MS.P.MALA
DESIGNATION:ASSISTANT PROFESSOR
UNIVERSITY NAME :PERIYAR MANIAMMAI
UNIVERSITY
DEPARTMENT OF BIOTECHNOLOGY

3. INTRODUCTION
• The DNA of the organism is used to create a genomic library.
• Is isolated from cells and then cut into specific-sized pieces using a restriction
enzyme. Using DNA ligase, the components are subsequently put into the vector.
A host immune system, such as a populace, may then occupy the vector DNA.
• E. Coli or yeast, with each cell having just one vector molecule.
• The employment of a host cell to carry the vector makes clone amplification and
library extraction for analysis much easier.

4. GENOMIC LIBRARY DEFINITION
• A genomic library is refer to a collection of
overlapping DNA fragments that together make up
the total genomic DNA of a single organism.
• The DNA is stored in a population of identical
vectors, each containing a different insert of DNA.

5. CDNA LIBRARY DEFINITION
• A cDNA library is a collection of cloned DNA sequences
that are complementary to the mRNA that was extracted
from an organism or tissue (the ‘c’ in cDNA stands for
‘complementary’).
• A combination of cloned cDNA (complementary DNA)
fragments inserted into a collection of host cells, which
constitute some portion of the transcriptome of the
organism and are stored as a “library”.

6. GENOMIC LIBRARY
CONSTRUCTION
• The Genomic Library or gene bank is constructed
by a shotgun experiment where the entire
genome of the cell is cloned in the form of
random and unidentifiable clones. It uses the
chain termination method (Sanger’s sequencing)
to sequence the DNA molecules. The DNA clones
are produced by the following steps:
• Isolating the DNA fragments that are to be cloned
and joining them with suitable vectors like the
lambda phage.
• Now, it is introduced into the host cell at high
efficiency to get a large number of independent
clones.
• Finally, the desired clones are selected and used
for the construction of the genomic library.

7. CDNA LIBRARY CONSTRUCTION
• Creation of a cDNA library starts with mRNA
instead of DNA. Messenger RNA carries
encoded information from DNA to ribosomes
for translation into protein.
• To create a cDNA library, these mRNA
molecules are treated with the enzyme
reverse transcriptase, which is used to make a
DNA copy of an mRNA (i.e., cDNA).
• A cDNA library represents a sampling of the
transcribed genes, but a genomic library
includes untranscribed regions.

8. GENOMIC DNA LIBRARY
• What it is : Collection of clones having
the complete genomic DNA of an
entity.
• What does it contains :Represents all
genes.
• Size :Vast in comparison to cDNA
library
• Coding and non-coding sequences
:Clone contains sequences seen on
mRNA only, not the complete gene.
• Collection of clones having
complementary DNA to the mRNA of
an entity.
• Represents genes expressed in a
particular cell at a given period of
time.
• Smaller compared to genomic DNA
library.
• Clone contains sequences seen on
mRNA only, not the complete gene.
CDNA LIBRARY
DIFFERENCE BETWEEN GENOMIC AND
CDNA LIBRARIES

9. GENOMIC DNA LIBRARY
• Important substance required for their
construction :Ligases and restriction
endonucleases
• Way greater number of recombinants
compared to cDNA library.
• DNA.
• Gene expression extracted from the
genomic library is challenging in the
prokaryotic systems, as they are
devoid of a splicing mechanism.
• Role in reverse transcriptase:Not
involved
• Vectors used : Cosmids, plasmids,
Lambda phage, YAC, BAC to harbour
larger fragments.
CDNA LIBRARY
• Reverse transcriptase enzyme
• How many recombinants to be
screened:: The number is way lesser in
comparison to the genomic DNA
library.
• Material required to start : mRNA
• Expression in prokaryotic system :Can
be directly expressed as it has only
coding sequences.
• Occurs in the synthesis of the first
cDNA strand.
• Phagemids, Plasmids, Lambda phage
for harbouring smaller fragments due
to the absence of introns

10. CONCLUSION
• Creating a genomic library A genomic library requires the production of
a huge number of recombinant DNA molecules.
• An organism’s genomic DNA is extracted and then digested using a
restriction enzyme.
• Repositories of raw material for use in genetic engineering.
• A large genome, such as that of humans, is most conveniently cloned
using vectors that can accommodate large fragments of DNA, such as
yeast artificial chromosomes, maintained in cell culture.

11. REFERENCE
• Losick, Richard; Watson, James D.; Tania A. Baker; Bell, Stephen; Gann,
Alexander; Levine, Michael W. (2008).
• Russell, David W.; Sambrook, Joseph (2001).
• Hartwell, Leland (2008).
• Muse, Spencer V.; Gibson, Greg (2004).
• Henry RJ, Edwards M, Waters DL, et al. (November 2012).
• Sanger F, Air GM, Barrell BG, et al. (February 1977).
• Cichon S, Mühleisen TW, Degenhardt FA, et al. (March 2011).

12. • Menon R, Farina C (2011).
• Yoo EY, Kim S, Kim JY, Kim BD (August 2001).
• Osoegawa K, de Jong PJ, Frengen E, Ioannou PA (May 2001).
• John R. McCarrey; Williams, Steven J.; Barton E. Slatko (2006).
• Peterson, Daniel; Jeffrey Tomkins; David Frisch (2000).
• Kim UJ, Birren BW, Slepak T, et al. (June 1996).
• Blaber, Michael. “Genomic Libraries”. Retrieved 1 April 2013.
• Fuesler J, Nagahama Y, Szulewski J, Mundorff J, Bireley S, Coren JS (April
2012).
• Pareek CS, Smoczynski R, Tretyn A (November 2011).
• Pennisi E (April 2003), Chen Y, Lin X, Yi Y, Lu Y, Zhang Z (2009).
• Ying, Shao-Yao (2004).
• P., Clark, David (2009).

13. MULTIPLE CHOICE QUESTIONS
• 1. Gene libraries made from genomic DNA are called genomic libraries and those made from
complementary DNA are known as cDNA libraries.
• A.TRUE. B.FALSE
• 2.This vector can be used to construct genomic libraries.
• A.Plasmid. B.Phage. C.Cosmid. D.BAC.
• 3.This enzyme is used to dephosphorylate the vector.
• A.Terminal transferase. B.Alkaline phosphatase. C) Reverse transcriptase. D)DNA ligase

14. • 4)The first genomic libraries were cloned in _______________
• a) Plasmid b) Bacteria c) Human d) Plants
• 5)Genomic library construction is concerned with ___________
• a) Gene isolation b) Protein production c) Antibiotics d) Regeneration
• 6)What is the approximate size of fragments given off by EcoR1?
• a) 1 kb b) 2 kb c) 3 kb d) 4 kb

15. • ANSWERS:
• 1) A
• 2) D
• 3) B
• 4) A
• 5) A
• 6) D