Editing CDNA library

{{Short description|Type of DNA library}}
{{lowercase|title=cDNA Library}}
{{More references|date=May 2008}}
A '''cDNA library''' is a combination of cloned cDNA ([[complementary DNA]]) fragments inserted into a collection of host cells, which constitute some portion of the [[transcriptome]] of the organism and are stored as a "[[DNA library|library]]". cDNA is produced from fully transcribed [[mRNA]] found in the [[Cell nucleus|nucleus]] and therefore contains only the expressed genes of an organism. Similarly, tissue-specific cDNA libraries can be produced. In [[eukaryotic]] cells the mature mRNA is already [[RNA splicing|spliced]], hence the cDNA produced lacks [[introns]] and can be readily expressed in a bacterial cell. 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 library|genomic DNA library]].<ref>{{Cite web |title=cDNA Libraries - an overview {{!}} ScienceDirect Topics |url=https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/cdna-libraries#:~:text=A%20cDNA%20library%20is%20a,stands%20for%20'complementary'). |access-date=2024-02-19 |website=www.sciencedirect.com}}</ref><ref>{{Cite web |title=cDNA Library Overview and Applications - Creative Biogene |url=https://www.creative-biogene.com/support/cdna-library-overview-and-applications.html |access-date=2024-10-17 |website=www.creative-biogene.com}}</ref>

== cDNA Library Construction ==
[[File:Formation of a cDNA Library.jpg|thumb|300 px|Formation of a cDNA library.]]

cDNA is created from a mature [[mRNA]] from a eukaryotic cell with the use of [[reverse transcriptase]]. In eukaryotes, a [[mRNA#Polyadenylation|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 (molecular biology)|primer]] site for reverse transcription. This has the problem that not all transcripts, such as those for the [[histone]], encode a [[poly-A tail]].<ref>{{Citation |title=cDNA library {{!}}{{!}} How cDNA library is constructed? {{!}}{{!}} What are DNA libraries used for? | date=27 August 2019 |url=https://www.youtube.com/watch?v=URtcaoNEIrI |access-date=2024-02-19 |language=en}}</ref><ref>{{Cite web |last=Tamang |first=Sanju |date=2024-06-30 |title=DNA Library (Genomic, cDNA): Types, Preparation, Uses |url=https://microbenotes.com/dna-library-gene-library/ |access-date=2024-10-17 |website=microbenotes.com |language=en-US}}</ref>

=== mRNA extraction ===
Firstly, mRNA template needs to be isolated for the creation of cDNA libraries. Since mRNA only contains exons, the integrity of the isolated mRNA should be considered so that the protein encoded can still be produced. Isolated mRNA should range from 500 bp to 8 kb.<ref name=":0">{{Cite journal |last=Ying |first=Shao-Yao |date=2004 |title=Complementary DNA Libraries: An Overview |url=http://link.springer.com/10.1385/MB:27:3:245 |journal=Molecular Biotechnology |language=en |volume=27 |issue=3 |pages=245–252 |doi=10.1385/MB:27:3:245 |pmid=15247497 |s2cid=25600775 |issn=1073-6085|url-access=subscription }}</ref>  Several methods exist for purifying RNA such as [[trizol]] extraction and [[column purification]]. Column purification can be done using oligomeric dT nucleotide coated resins, and features of mRNA such as having a poly-A tail can be exploited where only mRNA sequences containing said feature will bind. The desired mRNA bound to the column is then [[Elution|eluted]]. 

=== cDNA construction ===
Once mRNA is purified, an ''oligo-dT'' primer (a short sequence of deoxy-thymidine nucleotides) is bound to the poly-A tail of the RNA. The primer is required to initiate DNA synthesis by the enzyme [[reverse transcriptase]]. This results in the creation of RNA-DNA hybrids where a single strand of complementary DNA is bound to a strand of mRNA.<ref name=":1" /> To remove the mRNA, the [[Ribonuclease H|RNAse H]] enzyme is used to cleave the backbone of the mRNA and generate free 3'-OH groups, which is important for the replacement of mRNA with DNA.<ref name=":0" /> [[DNA polymerase]] I is then added, the cleaved RNA acts as a primer the DNA polymerase I can identify and initiate replacement of RNA nucleotides with those of DNA.<ref name=":0" /> This is provided by the {{chem name|sscDNA}} itself by coiling on itself at the 3' end, generating a ''[[hairpin loop]]''. The polymerase extends the 3'-OH end, and later the loop at 3' end is opened by the scissoring action of [[S1 nuclease|''S<sub>1</sub> nuclease'']]. [[Restriction endonucleases]] and [[DNA ligase]] are then used to [[cloning|clone]] the sequences into bacterial [[plasmid]]s.

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 cells. cDNA libraries are most useful in [[reverse genetics]] where the additional genomic information is of less use. Additionally, 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 help ensure the insert is truly a gene.<ref name=":1">{{Cite book|title=Biotechnology : applying the genetic revolution|last=P.|first=Clark, David|date=2009|publisher=Academic Press/Elsevier|others=Pazdernik, Nanette Jean.|isbn=9780121755522|location=Amsterdam|oclc=226038060}}</ref> 

=== cDNA Library vs. Genomic DNA Library ===
cDNA library lacks the non-coding and regulatory elements found in genomic DNA. [[Genomic DNA library|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. BamHI. 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 ''[[Escherichia coli|E. coli]]'' host cell for cloning.

==See also==
* [[Functional cloning]]

==References==
<references />

==External links==
* [https://web.archive.org/web/20181102155217/http://fantom.gsc.riken.jp/ Functional Annotation of the Mouse database] ([[FANTOM]])
* [https://web.archive.org/web/20010716073355/http://dwb.unl.edu/Teacher/NSF/C08/C08Links/www.dur.ac.uk/~dbl0www/Staff/Croy/cDNAfigs.htm examples of cDNA synthesis and cloning]
* [[doi:10.1007/978-1-4939-2392-2_12|Preparation of cDNA libraries for high-throughput RNA sequencing analysis of RNA 5′ ends]]

[[Category:Molecular biology]]
[[Category:DNA]]