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cDNA Library Construction Protocol

cDNA Library Construction Protocol

Complementary DNA (cDNA) is produced from a fully transcribed messenger ribonucleic acid (mRNA) that contains only the expressed genes of an organism. Clones of such DNA copies of mRNAs are called cDNA clones. A cDNA library is a combination of cloned cDNA fragments constituting some portion of the transcriptome of an organism which are inserted into many host cells. The mRNA is spliced before translation into protein in eukaryotic cells. The DNA synthesized from the spliced mRNA doesn't have non-coding regions or introns of the gene. Therefore, the protein under expression can be sequenced from the DNA which is the key advantage of cDNA cloning over genomic DNA cloning.

The Workflow of cDNA Library Construction

Creation of a cDNA library starts with mRNA instead of DNA. Messenger RNA carries encoded information from DNA to ribosomes for translation into protein. To create a cDNA library, these mRNA molecules are treated with the enzyme reverse transcriptase, which is used to make a DNA copy of an mRNA (i.e., cDNA). A cDNA library represents a sampling of the transcribed genes, but a genomic library includes untranscribed regions.

1. Isolation of mRNA

First of all, it involves the isolation of total mRNA from a cell type or tissue of interest. It may be desirable to remove highly abundant tRNAs and rRNAs which might otherwise constitute the majority of the final library to the detriment of the detection of low abundance RNAs. We routinely remove tRNAs and other small RNAs<200 nt using a Kit from Creative Biogene and remove rRNAs using magnetic bead-based depletion kits. The 3’ ends of eukaryotic mRNA are composed of a string of 50 -250 adenylate residues (poly A Tail) which makes the separation easy from the much more prevalent rRNAs and tRNAs in a cell extract through a column containing oligo-dTs tagged onto its matrix.

The number of desired mRNA can be increased by the following methods:

  • Chromatographic purification of mRNA through the oligo-dT column, which retains mRNA molecules, resulting in their enrichment.
  • Spinning down mRNA using density gradient centrifugation.
  • mRNA preparation from specialized cell types, such as chicken oviduct, erythrocytes, developing seeds,β cells of pancreas etc.

2. Synthesis of the first strand of cDNA

mRNA being single-stranded cannot be cloned and is not a substrate for DNA ligase. It is first converted into DNA prior to insertion into a suitable vector.
1) A short oligo (dT) primer is annealed to the Poly (A) tail on the mRNA.
2) Reverse transcriptase extends the 3´-end of the primer by mRNA molecule as a template producing a cDNA: mRNA hybrid.
3) The mRNA from the cDNA: mRNA hybrid can be removed by alkaline hydrolysis or RNase H to give a single stranded (ss)-cDNA molecule.

Note: No primer is required because the 3´end of this ss-cDNA serves as its own primer generating a short hairpin loop at this end. The free 3´-OH is required for the synthesis of its complementary strand.

3. The second strand of cDNA generation

The ss-cDNA is converted into double stranded (ds) cDNA by either RTase or E. coli DNA polymerase. (It is essential to use only the minimal number of amplification cycles needed to obtain sufficient material for sequencing to avoid over-amplification of the libraries, which is a major source of bias in the results.)

4. Incorporation of cDNA into a vector

The ds-cDNA can be trimmed with S1 nuclease to obtain blunt–ended ds-cDNA molecule followed by addition of terminal transferase to tail the cDNA with C's and ligation into a vector. Because the blunt-end ligation is inefficient, short restriction-site linkers are first ligated to both ends.

5. Cloning of cDNAs

cDNAs are commonly cloned in phage insertion vectors. Bacteriophage vectors possess the following advantageous over plasmid vectors:

  • Are more desirable when a large number of recombinants are required for cloning low-abundant mRNAs as recombinant phages are produced by in vitro packaging.
  • Can easily handle and store large numbers of phage clones, as compared to the bacterial colonies carrying plasmids.

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