VPB 321: Animal Biotechnology (2+1)

PREPARATION OF cDNA LIBRARY

• cDNA is created from a mature mRNA from a eukaryotic cell with the use of an enzyme known as 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.

mRNA extraction

• 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 will 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-thymine 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 help 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 hair pin 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.