Editing CDNA library (section)

== 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.