cDNA Library
• cDNA is the double stranded complement of mRNA.
• It represents only transcribed genes i.e., DNA copies of the
RNA sequence (mRNA) and clone them.
• cDNA can be synthesized from mRNA by reverse transcriptase
enzyme.
• cDNA library refers to the collection of complementary DNA
prepared from mRNA.
• cDNA molecules to be cloned are only a few kilo base long and
conveniently inserted in plasmid cloning vectors.
Construction of cDNA Library:
1. synthesis of cDNA from mRNA
2. cloning of cDNA in vector
3. Screening of cdna library
cDNA preparation :
3 methods:
1.RNase H method
2.Self priming method.
3.Tailing and priming method.
(1) cDNA preparation by RNase H method:
• This approach involves the synthesis of complementary DNA
strand by reverse transcription to make an RNA: DNA duplex.
• The cDNA synthesis requires separation of polyadenylated
RNA (mRNA) from other RNA.
• This can be accomplished by fractionation of RNA on oligo-dT
cellulose in which short deoxy T residue have been covalently
attached via hydroxyl groups of the cellulose. mRNA is
separated from rest of the RNA.
• This is accomplished by passing a solution containing RNA
through a column of oligo-dT cellulose.
• The poly A tail of the RNA forms hydrogen bond with oligo-
dT, and poly A + RNA (mRNA) retains in the column. After
washing all other brand RNA from the column, the poly A +
RNA is eluted with low-salt buffer.
• The purified mRNA is mixed with reaction mixture containing
reverse transcriptase and four deoxyribonucleotides(dNTP’s)
• The first step in cDNA synthesis is the annealing of
chemically-synthesized oligo-dT primer to the 3′ poly A tail of
the RNA. Addition of 10-15 residue long primers initiates
synthesis of first DNA strand with reverse transcriptase and
dNTPs. As a result RNA: DNA duplex is formed. In the next
step, RNA strand is replaced by DNA strand.
• This process can be initiated by adding a low concentration of
RNase H together with DNA polymerase and dNTPs.
• Treatment of RNA: DNA duplex with RNase H result in the
nicking of RNA and produced free of 3′- hydroxyl groups.
• This acts as primers at 3′ end to initiate DNA synthesis and
finally DNA duplex strand is produced.
Disadvantage:
• Method depends on polyadenylation of mRNA.
• Very difficult to construct full length cDNA.
(2)cDNA synthesis by self-priming approach:
• In the second approach, once DNA: RNA duplex is secured, it
is then subjected to treatment with dilute sodium hydroxide
for alkaline hydrolysis of RNA leaving single DNA strand.
• Since oligo-dT cannot facilitate second strand synthesis, self-
priming takes place by chance occurrence of complementary
sequence between region near 3′-end and region to 5′-end
which consequently attains hair pin loop.
• The 3’OH region acts as a primer for the synthesis of second
DNA strand. Presence of loop is removed by treatment with SI
nuclease, which degrades single-strand regions (loops) and
subsequently results in the formation of double-stranded
blunt-ended DNA molecule .
• The generation of blunt ended cDNA molecule is then attached
with linkers before inserting them into the vector.
(3)TAILING AND PRIMING METHOD:
Steps:
• Reverse transcriptase enzyme synthesize cDNA complementary
to mRNA.
• Terminal transferase enzyme add C nucleotides specifically to
the3’ end.
• Alkaline hydrolyses is carried out to degrade RNA.
• Oligo dG primer is added to the resulting single strand and this
act as the primer for the 2ndstrand synthesis.
• DNA synthesis is initiated by the action of DNA polymerase and
dNTP’s.
SYNTHESIS OF COMPLEMENTARY DNA (cDNA)
Cloning of cDNA:
Phage vectors are prefered for cDNA cloning.
3 methods are used to clone cDNA.
1. Blunt end ligation
2. Use of linkers and adaptors
3. Homopolymer tailing
Screening Libraries:
Colony hybridization:
• A common method of screening plasmid-based genomic
libraries is to carry out a colony hybridization experiment.
• The protocol is similar for phage-based libraries except that
bacteriophage plaques, not bacterial colonies, are screened.
• In a typical experiment, host bacteria containing either a
plasmid based or bacteriophage-based library are plated out
on a petri dish and allowed to grow overnight to form colonies
(or in the case of phage libraries, plaques).
• A replica of the bacterial colonies (or plaques) is then obtained
by overlaying the plate with a nitrocellulose disc.
• The disc is removed, treated with alkali to dissociate bound
DNA duplexes into single-stranded DNA, dried, and placed in
a sealed bag with labelled probe.
• If the probe DNA is duplex DNA, it must be denatured by
heating at 80°C.
• The probe and target DNA complementary sequences must be
in a single stranded form if they are to hybridize with one
another.
• Any DNA sequences complementary to probe DNA will be
revealed by autoradiography of the nitrocellulose disc.
• Bacterial colonies (phage plaques) containing clones bearing
target DNA are identified on the film and can be recovered
from the master plate.
Immunological Screening:
This is another novel method of screening a gene library. This
method can be used as an alternative to DNA probe. The cells are
allowed to transcribe and translate and the presence of protein can
be identified by corresponding antibody molecule.
The technique is described as follows:
(i) Discrete colonies are formed on the master plate,
(ii) Transfer sample of cells from each colony to nitrocellulose or
nylon membrane.
(iii) Allow the cells to transcribe and translate. The cells are then
lysed and proteins are bound to matrix.
(iv) Initially primary antibody treatment is done to faciliate the
binding of primary antibody to protein molecule bound on the solid
matrix.
(v) Add secondary antibody that binds only to the primary antibody.
Colorimetric reaction takes place only if the secondary antibody is
present (Enzyme bound secondary antibody develops coloured
compound when treated with respective substrate).
(vi) A colony on the master plate that corresponds to a positive
response on the matrix is identified. The cells from the positive
colony on the master plate are sub-cultured as this may carry insert
DNA that encodes the protein that bind primary antibody .
3. OLIGONUCLEOTIDE SCREENING
Oligonucleotide probes are used to detect proteins.
