Priyanshi mishra (3)

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Dr. Harisingh gour central university sagar
(M.P.)
GENETIC ENGINEERING Open elective
course, Course code - Zoo E 229A
2nd Mid semester
cDna Library
Presented to- Dr. Rashmi Shrivastava,
Dr. Rajkumar kori Department of zoology.
Presented by- Name - Priyanshi mishra, Roll
number - Y20264010 Department- Microbiology,
Class - Msc 2nd semester
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Gene libraries
Collection of different DNA sequences from an
organism, each of which has been cloned into a
vector for ease of purification, storage and
analysis.
Genomic library (made from genomic DNA) Whole
genes/promoters and introns (eukaryotes)/ operons
(bacteria)/DNA regulatory elements.
Gene Libraries
cDNA library (made from cDNA copy of mRNA) mRNA
transcripts only/ 5 and 3 untranslated regions
(UTRs)/ no introns/ tissue specific.
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cDNA library
mRNA
cDNA library represents the population of mRNAs,
it only contains the exon of protein structural
gene.
Reverse transcriptase
cDNA
Replication
dscDNA
Vector
Recombinant DNA
E.coli
Recombinant DNA in E.coli
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Steps in construction of cDNA library
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1) Isolation of mRNA
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(2)
Prepration of complementary DNA strand
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Synthesis of first and second strand of cDNA
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3) Incorporation of cDNA into a vector
The ds-cDNA can be trimmed with S1 nuclease to
obtain blunt ended ds-cDNA molecule followed by
addition of terminal transferase to tail the cDNA
with Cs and ligation into a vector. Because the
blunt end ligation is inefficient, short
restriction-site linkers are first ligated to
both ends.
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Modification of cDNA termini
using linkers.
Action of methylases
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4) Cloning of cDNA
cDNAs are usually cloned in phage insertion
vectors. Bacteriophage vectors offer the
following advantageous over plasmid vectors,  
are more suitable when a large number of
recombinants are required for cloning
low-abundant mRNAs as recombinant phages are
produced by in vitro packaging.   can easily
store and handle large numbers of phage clones,
as compared to the bacterial colonies carrying
plasmids. Plasmid vectors are used extensively
for cDNA cloning, particularly in the isolation
of the desired cDNA sequence involving the
screening of a relatively small number of clones.
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Construction of cDNA library
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Application of cDNA library

• To Clone and express eukaryotic genes in
  prokaryotes.
• To express specific protein whenever needed.
• In vitro study of gene function.
• Carcinogens identification.
• Useful for isolating gene that codes for
  particular mRNA.
• Cloning of full length cDNA molecules for in
  vitro study of gene function.
• To obtain pure sample of a gene.
• To get high yield of recombinant DNA.
• Commercial production of protein and other
  biological molecules.
• Study of alternative splicing in different cells
  or tissues.
• To study the difference in protein expression
  between two different cells.

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