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cDNA Library

The document discusses the construction and applications of a cDNA library. Key points include: - A cDNA library contains complementary DNA clones synthesized from mRNA of a particular tissue. - Construction involves isolating mRNA, synthesizing cDNA, cloning cDNA into a vector, and screening clones. - Applications include gene discovery, expression analysis, and isolating genes expressed in a given tissue.

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282 views

cDNA Library

The document discusses the construction and applications of a cDNA library. Key points include: - A cDNA library contains complementary DNA clones synthesized from mRNA of a particular tissue. - Construction involves isolating mRNA, synthesizing cDNA, cloning cDNA into a vector, and screening clones. - Applications include gene discovery, expression analysis, and isolating genes expressed in a given tissue.

Uploaded by

janapriya
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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MANIKALA.

D
19D1751

CDNA LIBRARY

MANIKALA.D

CDNA LIBRARY 19D1751


INTRODUCTION:

DNA library
 A DNA library is a collection of DNA clones, gathered together as a source of
DNA for sequencing, gene discovery, or gene function studies.
 There are two types of DNA libraries: cDNA and Genomic
cDNA Library
• A cDNA library is a set of cDNA clones prepared from the mRNAs isolated
from a particular type of tissue.•
 The cDNA library contains only complementary DNA molecules synthesized
from mRNA molecules in a cell.
 This molecules represents all the gene that are expressed in the cell at different
stage of its development.
CDNA LIBRARY – AN OVERVIEW
CONSTRUCTION OF CDNA LIBRARY:

 1. Isolation of mRNA from cell & synthesis of cDNA from this eukaryotic
functional mRNA
 2. Cloning of synthesized cDNAs in suitable vector like plasmid or A phage.
 3. Introduction to suitable host like bacterium E.coli
 4. Screening & maintenance of set of clones containing cDNA
mRNA Extraction:
 Firstly, the mRNA is obtained and purified from the rest of the RNA’s. Several
methods exist for purifying RNA such as trizol extraction and column purification.
 Column purification is done by using oligomeric dT nucleotide coated resins where
only the mRNA having the poly-A tail will bind.
 The mRNA is eluted by using eluting buffer and some heat to separate the mRNA
strands from oligo-dT.
CDNA CONSTRUCTION:

 Once mRNA is purified, oligo-dT (a short sequence of deoxy-thymidine nucleotides) is tagged as a


complementary primer which binds to the poly-A tail providing a free 3¹-OH end that can be
extended by reverse transcriptase to create the complementary DNA strand.
 Now, the mRNA is removed by using a RNAse enzyme leaving a single stranded cDNA (sscDNA).
This sscDNA is converted into a double stranded DNA with the help of DNA polymerase. However,
for DNA polymerase to synthesize a complementary strand a free 3'-OH end is needed.
 This is provided by the sscDNA itself by generating a hairpin loop at the 3' end by coiling on itself.
 The polymerase extends the 3'-OH end and later the loop at 3' end is opened by the scissoring action
of S₁ nuclease. Restriction endonucleases and DNA ligase are then used to clone the sequences into
bacterial plasmids.
 The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once
selected, stocks of the bacteria are created which can later be grown and sequenced to compile the
cDNA library.
SCREENING:

 • A probe is a piece of DNA or RNA used to detect specific nucleic


acid sequences by hybridization (binding of two nucleic acid chains
by base pairing).
 Oligonucleotide can be used as a probe.• They are radioactively
labeled so that the hybridized nucleic acid can be identified by
autoradiography.
 Two general approaches are available for screening libraries to
identify clones carrying a gene or other DNA region of interest:
(1) Detection with oligonucleotide probes that bind to the clone of
interest and
(2)Detection based on expression of the encoded protein
APPLICATIONS OF CDNA LIBRARY:

 Storage of reduced amount of information due to the removal of non-coding regions.


 cDNA can be directly expressed in prokaryotic organisms.
 cDNA libraries are useful in reverse genetics where the additional genomic information is of less use.
 cDNA library is useful for isolating gene that codes for particular mRNA.
 cDNA library is a powerful and useful tool in the area of biotechnology.
 It is helpful in expressing eukaryotic genes in prokaryotes, which helps in the transcription process of
prokaryotes. It is used to isolate DNA sequences to code mRNA.
 Advantage of cDNA library is to isolate homologous genes.
 It is also used for the screening genomic libraries to isolate specific cDNA.
 cDNA of proteins can facilitate to generate antibodies and monoclonal antibodies.
 The most important application of cDNA library is to study expression of mRNA.
DISADVANTAGES OF CDNA LIBRARY:

 The disadvantage of a cDNA library is that it contains only sequences


that are present in mature mRNA.
 Introns and any other sequences that are altered after transcription are
not present; sequences, such as promoters and enhancers that are not
transcribed into RNA also are not present in a cDNA library
 It is also important to note that the cDNA library represents only those
gene sequences expressed in the tissue from which the RNA was isolated
 Furthermore, the frequency of a particular DNA sequence in a cDNA
library depends on the abundance of the corresponding mRNA in the
given tissue.
CLONING OF CDNA LIBRARY:

 cDNA molecules can be cloned by using restriction site linkers. Linkers are short,
double stranded pieces of DNA (oligodeoxyribonucleotide) about 8 to 12 nucleotide
pairs long that include a restriction endonuclease cleavage site e g. BamHl.
 Both the cDNA and the linker have blunt ends which can be ligated together using a
high concentration of T4 DNA ligase.
 Then sticky ends are produced in the cDNA molecule by cleaving the cDNA ends
(which now have linkers with an incorporated site) with the appropriate
endonuclease.
 A cloning vector (plasmid) is then also cleaved with the appropriate
endonuclease.Following "sticky end" ligation of the insert into the vector the
resulting recombinant DNA molecule is transferred into E.coli host cell for cloning.

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