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cDNA Library - Wikipedia

A cDNA library contains cloned cDNA fragments from an organism's transcriptome inserted into host cells. cDNA is produced from mRNA and lacks introns, containing only expressed genes. Tissue-specific cDNA libraries provide information about gene expression in particular tissues. While cDNA libraries are useful for identifying gene products, they lack regulatory elements found in genomic DNA libraries.

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302 views4 pages

cDNA Library - Wikipedia

A cDNA library contains cloned cDNA fragments from an organism's transcriptome inserted into host cells. cDNA is produced from mRNA and lacks introns, containing only expressed genes. Tissue-specific cDNA libraries provide information about gene expression in particular tissues. While cDNA libraries are useful for identifying gene products, they lack regulatory elements found in genomic DNA libraries.

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cDNA library

A cDNA library is a combination of cloned cDNA (complementary DNA) fragments inserted into a col lection of
host cel ls, which constitute some portion of the transcriptome of the organism and are stored as a "library".
cDNA is produced from ful ly transcribed mRNA found in the nucleus and therefore contains only the
expressed genes of an organism. Similarly, tissue-specific cDNA libraries can be produced. In eukaryotic
cel ls the mature mRNA is already spliced, hence the cDNA produced lacks introns and can be readily
expressed in a bacterial cel l. While information in cDNA libraries is a powerful and useful tool since gene
products are easily identified, the libraries lack information about enhancers, introns, and other regulatory
elements found in a genomic DNA library.

cDNA Library Construction

Formation of a cDNA library.


cDNA is created from a mature mRNA from a eukaryotic cel l with the use of reverse transcriptase. In
eukaryotes, a poly-(A) tail (consisting of a long sequence of adenine nucleotides) distinguishes mRNA from
tRNA and rRNA and can therefore be used as a primer site for reverse transcription. This has the problem
that not al l transcripts, such as those for the histone, encode a poly-A tail.

mRNA extraction

Firstly, the mRNA is obtained and purified from the rest of the RNAs. 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 wil l bind. The rest of the RNAs are
eluted out. 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 3'-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 hel p 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.

cDNA Library uses

cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is
reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are used to
express eukaryotic genes in prokaryotes. Prokaryotes do not have introns in their DNA and therefore do not
possess any enzymes that can cut it out during transcription process. cDNA does not have introns and
therefore can be expressed in prokaryotic cel ls. cDNA libraries are most useful in reverse genetics where the
additional genomic information is of less use. Additional ly, cDNA libraries are frequently used in functional
cloning to identify genes based on the encoded protein's function. When studying eukaryotic DNA, expression
libraries are constructed using complementary DNA (cDNA) to hel p ensure the insert is truly a gene.[1]
cDNA Library vs. Genomic DNA Library

cDNA library lacks the non-coding and regulatory elements found in genomic DNA. Genomic DNA libraries
provide more detailed information about the organism, but are more resource-intensive to generate and
keep.

Cloning of cDNA

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. BamH I. 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. Fol lowing "sticky end" ligation of the
insert into the vector the resulting recombinant DNA molecule is transferred into E. coli host cel l for cloning.

See also

Functional cloning

References

1. P., Clark, David (2009). Biotechnology : applying the genetic revolution. Pazdernik, Nanette Jean.
Amsterdam: Academic Press/Elsevier. ISBN 9780121755522. OCLC 226038060 (https://www.worldca
t.org/oclc/226038060) .

External links

Functional Annotation of the Mouse database (https://web.archive.org/web/20181102155217/http://fanto


m.gsc.riken.jp/) (FANTOM)

examples of cDNA synthesis and cloning (https://web.archive.org/web/20010716073355/http://dwb.unl.e


du/Teacher/NSF/C08/C08Links/www.dur.ac.uk/~d bl0www/Staff/Croy/cDNAfigs.htm)
Retrieved from "https://en.wikipedia.org/w/index.php?
title=CDNA_library&oldid=955474526"


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