This document provides information about synthesizing and cloning cDNA from mRNA. It describes how cDNA libraries are constructed by isolating mRNA, synthesizing cDNA via reverse transcription, treating cDNA ends, ligating the cDNA to vectors, and transforming the vectors into host cells. The key steps include mRNA purification, first and second strand cDNA synthesis, linker ligation, vector ligation, and transformation. cDNA libraries are useful for eukaryotic gene analysis as they contain only expressed genes without introns.

1. 5/26/2014 PBT 505 Techniques in Molecular Biology-2
{0+2} Deptt. of Plant Biotechnology
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2. COURSE TITLE:- TECHNIQUES IN MOLECULAR BIOLOGY-2
COURSE NO:-PBT 509(0+2)
TOPIC: SYNTHESIS AND CLONING OF cDNA
COURSE TEACHER-
Dr.K.M.Harini kumar
PROFFESOR
DEPT OF PLANT BIOTECHNOLOGY
PRESENTED BY-
BHARATI.G.S.
I.D. NO- PALB9299
DEPT. OF PLANT BIOTECHNOLGY
UAS,GKVK,BANGLORE.
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3. Gene library: a collection of different DNA
sequence from an organism, each of which
has been cloned into a vector for ease of
purification, storage and analysis.
Gene library
Genomic libraries
(made from genomic DNA)
cDNA libraries
(made from cDNA- copy of mRNA
I1 Genomiclibraries
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5. What is cDNA library?
cDNAlibrary is a combination of cloned
cDNA (complementary DNA) fragments
inserted into a collection of host cells,
which together constitute some portion
of the transcriptome of the organism.
 cDNA is produced from fully
transcribed mRNA found in the nucleus
and therefore contains only the
expressed genes of an organism.
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6. cDNA libraries:-
The cDNA library
represents the
population of
mRNAs, it only
contains the exons
of protein’s
structural genes.
mRNA
Reverse transcripta
cDNA
replication
dscDNA
vector
recombinate DNA
E. coli
recombinate DNA in E.coli
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7. Importance of cDNA libraries
1. No cDNA library was made
from prokaryotic mRNA.
•
•
Prokaryotic mRNA is very unstable
Genomic libraries of prokaryotes
are easier to make and contain all
the genome sequences.
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8. 2) cDNA libraries are very useful for
eukaryotic gene analysis
Condensed protein encoded, gene libraries
,have much less junk sequences.
 cDNAs have no introns so genes can be
expressed in E.coli directly.
Are very useful to identify new genes.
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9. Construction cDNA libraries:-
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12. Characteristics of cDNA libraries:-
Reverse transcription of mRNA
Dependent on gene expression
No introns
Expression is feasible if linked to a suitable
promoter
Useful for analysis of coding regions and
gene functions
No cDNA library was made from
prokaryotic mRNA because prokaryotic.
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13. mRNA are very unstable and easy to make
genomic library.
• cDNA libraries are very useful for
eukaryotic gene analysis because
condensed protein encoded gene library
have much less junk sequences.
• Very useful to identify genes.
• Tissue or cell specific.
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14. mRNA isolation, purification
Check theRNA integrity
Fractionate and enrich mRNA
Synthesis of cDNA
Treatment of cDNA ends
Ligation to vector 14
Synthesis of cDNA:-

15. mRNA isolation
•Most eukaryotic mRNAs are polyadenylated at
their 3’ ends
•oligo (dT) can be bound to the poly(A) tail
and used to recover the mRNA.
AAAAAAAAAAn5’ cap
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16. Three methods to isolate mRNA.
1.Traditionally method was done by pass a
preparation of total RNA down a column of
oligo (dT)-cellulose
2.More rapid procedure is to add oligo(dT)
linked to magnetic beads directly to a cell
lysate and ‘pulling out’ the mRNA using a
strong magnet
3.Alternative route of isolating mRNA is
lysing cells and then preparing mRNA-
ribosome complexes on sucrose gradients
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17. Make sure that the mRNA is not
degraded.
Methods:
Translating the mRNA : use cell-free
translation system as wheat germ extract or
rabbit reticulocyte lysate to see if the mRNAs
can be translated
Analysis the mRNAs by gel
elctrophoresis: use agarose or
polyacrylamide gels
Check the mRNA integrity
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18. Cloning the particular mRNAs
Is useful especially one is trying to clone a
particular gene rather to make a complete
cDNA library.
Fractionate on the gel: performed on
the basis of size, mRNAs of the interested
sizes are recovered from agarose gels
Enrichment: carried out by hybridization
Example: clone the hormone induced mRNAs
(substrated cDNA library)
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19. Synthesis of cDNA :
First stand synthesis: materials as
reverse transcriptase ,primer( oligo(dT) or
hexanucleotides) and dNTPs
Second strand synthesis: best way of
making full-length cDNA is to ‘tail’ the 3’-
end of the first strand and then use a
complementary primer
to make the second.
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20. 5’ mRNA AAAAA-3’
HO-TTTTTP-5’
Reverse transcriptase
Four dNTPs
TTTTTP-5’
5’ mRNA AAAAA-3’
cDNA
cDNA
cDNA
TTTTTP-5’
3’
3’-CCCCCCC TTTTTP-5’
Terminal transferase
Alkali (hydrolyaes RNA)
Purify DNA oligo(dG)
Klenow polymerase or reverse
Transcriotase Four dNTPs
dCTP
5’ mRNA AAAAA-3’
5’-pGGGG-OH
3’-CCCCCCC
5’-pGGGG
3’-CCCCCCC TTTTTP-5’
-3’
Duplex cDNA
The first strand synthesis 20

21. 5’-pGGGG
3’-CCCCCCC
3’-GGCTTAAGCCCCCC
5’-pAATTCGGGGGG
TTTTTGGCTTAAGCC-OH
CCGAATTCGG-3’
3’-CCCC
3’-CCCCCCC
5’-pGGGG
5’-pGGGG
TTTTTp-5’
-3’
TTTTTp-5’
-3’
-3’
TTTTTGGCTTAAp-5’
HO-CCG/AATTCGG-3’
3’-GGCTTAA/GCC-OH
CCG-3’
Duplex cDNA
Single strand-specific nuclease
3’-CCC TTTTTp-5’
Klenow polymerase
treat with E.coRI methylase
Add E.colRI linkers
using T4 DNA ligase
HO-CCGAATTCGGGGGG
E.colRI digestion
Ligate to vector and transfom
Second strand synthesis 21

22. Treatment of cDNA ends
Blunt and ligation of large fragment is not
efficient, so we have to use special acid linkers to
create sticky ends for cloning.
The process :
Move protruding 3’-ends(strand-special nuclease)
Fill in missing 3’ nucleotide (klenow fragment of
DNA polyI and 4 dNTPs)
Ligate the blunt-end and linkers(T4 DNA ligase)
Restriction enzyme digestion (E.coRI )
Tailing with terminal transferase or
using adaptor molecules
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23. Ligation to vector
Any vectors with an E.coRI site would suitable
for cloning the cDNA.
The process :
Dephosphorylate the vector with alkaline
phosphatase
Ligate vector and cDNA with T4 DNA ligase
(plasmid or λ phage vector)
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24. Vectors
According to genome’s size,we can select a
proper vector to construct a library .
Vectors Plasmid
insert (kb) 5
phageλ
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cosmid YAC
45 1000
The most commonly chosen genomic cloning vectors
are λ relacement vectors which must be digested with
restriction enzymes to produce the two λ end fragment
or λ arms between which the genomic DNA will be
digested
I1 Genomiclibraries
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25. cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
λ phage vector in cloning
cos cos
Long (left)
arm
short (right)
arm
Exogenous DNA
(~20-23 kb)
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26. Linkers for Cloning DNA
• Any DNA fragment can have a
specific restriction site added to
the ends by ligating on a
“linker”.
• Linkers are small, synthetic
(made in the lab, or ordered
from a company) DNA
fragments which contain the
recognition sequence for one
or more restriction enzymes.
• After ligating on linkers, the
DNA is cut with the
appropriate restriction enzyme
to produce ends for cloning.
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27. Hybridization:-
• The idea is that if DNA is made single stranded
(melted), it will pair up with another DNA (or RNA)
with the complementary sequence. If one of the DNA
molecules is labeled, you can detect the hybridization.
• Basic applications:
• Southern blot: DNA digested by a restriction enzyme then
separated on an electrophoresis gel
• Northern blot: uses RNA on the gel instead of DNA
• Colony hybridization: detection of clones
• Microarrays
• Polymerase Chain Reaction
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28. Southern Blot:-
• Used to detect a specific
DNA sequence in a
complex mixture, such as
genomic DNA
• Cut DNA with restriction
enzyme, then run on an
electrophoresis gel.
• Suck buffer through the gel
into a nitrocellulose
membrane. The buffer goes
through but the DNA sticks
to the membrane.
• Fix the DNA to the
membrane
permanently with UV
or heat
• Hybridize membrane to a
radioactive probe, then
detect specific bands with
autoradiography.
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29. Colony Hybridization:-
Bacterial colonies (or
phage plaques) containing
recombinant DNA are
grown on agar, then blotted
to nitrocellulose and
hybridized as with probe.
The colonies on the agar
plates stay alive, and once
the correct colony has been
detected, it can be picked
and grown up for further
work.
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In Situ Hybridization:-
• Using tissues or tissue
sections.
• Often done with non-
radioactive probes because
the high energy of 32P
emission gives an imprecise
view of hybridization.
• Counterstain the tissue so
non-hybridizing parts are
visible.
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31. Microarrays:-
• Place probes from many
different genes on a glass
microscope slide, then
hybridize to cDNA made
from messenger RNA
isolated from a tissue. we
see which genes are active
in that tissue.
• Mostly done with 60mers:
60 bases long, synthetic
oligonucleotides, made
using sequence
information from the
genes.
• cDNA is fluorescently labeled
• Often 2 conditions are
compared (control and
experimental), using red
and green fluorescent tags.
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32. Polymerase Chain Reaction:-
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• Another way to get our gene and it is very common.
• Based on a knowledge of the DNA sequence of a piece
of DNA.
• Allows we have to design primers that, along with a
thermostable DNA polymerase, let’s make all the
DNA that we need.

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36. Advantages of cDNA:-
• cDNA library is a collection of actively expressed genes
in the cells or tissue from which the mRNA was
isolated.
• We will get the expressed genes.
• Introns are not cloned in a cDNA library, which greatly
reduces the total amount of DNA that is cloned
compared to a genomic library.
• it isolate homologous genes.
• cDNA of proteins can facilitate to generate antibodies
and monoclonal antibodies.
• The most important application of cDNA library is to
study expression of mRNA.
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37. Disadvantages of cDNA:-
• One disadvantage is that cDNA libraries can be difficult
to create and screen if a source tissue with an
abundant amount of mRNA for the gene is not
available.
• we don't get control sequences or introns, and
frequency depends on level of expression.
• First strand synthesis often does not go to completion.
• Individual cDNA clones will frequently have the reverse
complement of only part of the mRNA.
• Multiple cDNA clones from a single mRNA will be
present in the library.
• Priming second strand synthesis is inefficient.
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38. Application of cDNA library:-
• Discovery of novel genes.
• Elucidation of gene function.
• In vitro study of gene function.
• Toobtain pure sample of a gene.
• Toget high yields of recombinant cDNA.
• Commercial production of proteins and other
biological molecules.
• Study the alternative splicing.
• Carcinogen identification.
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39. REFERENCES
Biotechnology expanding horizon by B.D.Singh
Biotechnology by P.K.Gupta
• Cloning and characterization of two Argonaute genes in
wheat (Triticum aestivum L.) by Fanrong Meng, Haiying Jia, Na
Ling, Yinlei Xue, Hao Liu, Ketao Wang, Jun Yin and Yongchun.
“BioMed Central “.
• Cloning and characterization of TaSnRK2.3, a novel SnRK2
gene in common wheat by Shanjun Tian, Xinguo Mao,
Hongying Zhang, Shuangshuang Chen,Chaochao Zhai, Shimin
Yang and Ruilian Jing. ” Journal of Experimental Botony”.
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