cDNA Libraries

cDNA Libraries:

  • It represents only transcribed genes i.e., DNA copies of the RNA sequence (mRNA) and clone them. 
  • Libraries of these types are highly feasible as they represent not only an expressed sequence of the gene, but also the sequences produced after post-transcriptional modification such as removal of introns etc.
  •  Besides, cDNA molecules to be cloned are only a few kilobase long and conveniently inserted in plasmid cloning vectors.
  • However, viral lambda phage vector is preferred due to subsequent screening process:

(i) cDNA preparation by RNase H method:

  • This approach involves the synthesis of complementary DNA strands by reverse tran­scription to make an RNA: DNA duplex. 
  • The cDNA synthesis requires separation of polyadenylated RNA (mRNA) from other RNA. 
  • This can be accomplished by fractionation of RNA on oligo-dT cellulose in which short deoxy T residues have been covalently attached via hydroxyl groups of the cellulose. 
  • mRNA is separated from the rest of the RNA.
  • This is accomplished by passing a solution containing RNA through a column of oligo-dT cellulose. 
  • The poly A tail of the RNA forms hydrogen bond with oligo-dT, and poly A + RNA (mRNA) retains in the column. 
  • After washing all other brand RNA from the column, the poly A + RNA is eluted with a low-salt buffer. 
  • The purified mRNA is mixed with a reaction mixture containing reverse transcription and four deoxyribonucleotides.
  • The first step in cDNA synthesis is the annealing of chemically-synthesized oligo-dT primer to the 3′ poly A tail of the RNA.
  •  Addition of 10-15 residue long primers initiates synthe­sis of the first DNA strand with reverse transcriptase and dNTPs. 
  • As a result RNA: DNA duplex is formed. 
  • In the next step, RNA strand is replaced by DNA strand.
  • This process can be initiated by adding a low concentration of RNase H together with DNA polymerase and dNTPs. 
  • Treatments of RNA: DNA duplex with RNase H result in the nicking of RNA and produced free of 3′- hydroxyl groups.
  • This acts as primers at 3′ end to initiate DNA synthesis. 
  • As DNA chains are synthesised, any molecule that is base-paired to the DNA template further down are degraded and leaves DNA duplex strand.

(ii) cDNA synthesis by self-priming approach:

  • In the second approach, once DNA: RNA duplex is secured, it is then subjected to treat­ment with dilute sodium hydroxide for alkaline hydrolysis of RNA leaving a single DNA strand. 
  • Since oligo-dT cannot facilitate second strand synthesis, self-priming takes place by chance occurrence of complementary sequence between region near 3′-end and region to 5′-end which consequently attains hair pin loop.
  • The base paired region acts as a primer for the synthesis of the second DNA strand. 
  • Presence of loop is removed by treatment with SI nuclease, which de­grades single-strand regions (loops) and subsequently results in the formation of double-stranded blunt-ended DNA molecules.
  • The generation of blunt ended cDNA molecules is then attached with linkers before inserting them into the vector.
Print Friendly, PDF & Email

Leave a Reply