User:Biologystudent4/CDNA library
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[edit]cDNA is created from a mature mRNA from a eukaryotic cell 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 all transcripts, such as those for the histone, encode a poly-A tail.
mRNA extraction[edit]
[edit]Firstly, mRNA template needs to be isolated for the creation of cDNA libraries. mRNA only contains exons which are the regions of gene that encode protein. 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. 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 eluted.
cDNA construction[edit]
[edit]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. To remove the mRNA, the 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. 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. This is provided by the 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. 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.