Illustrate the steps of constructing a recombinant c-DNA from an m-RNA.

Steps in Constructing Recombinant cDNA from mRNA

The construction of recombinant cDNA involves a series of enzymatic reactions and molecular biology techniques. Here's a detailed breakdown of the process:

1. mRNA Isolation and Purification

The process begins with isolating mRNA from the source cells or tissues. This is typically achieved using oligo(dT) cellulose chromatography. mRNA possesses a poly(A) tail at its 3’ end, which selectively binds to the oligo(dT) column, allowing for its purification from other RNA species like rRNA and tRNA.

2. First Strand cDNA Synthesis

Once mRNA is purified, it serves as a template for the enzyme reverse transcriptase. This enzyme, originally discovered in retroviruses, synthesizes a complementary DNA strand (first-strand cDNA) using the mRNA as a template. This reaction requires a primer, typically an oligo(dT) primer that anneals to the poly(A) tail of the mRNA, or a random hexamer primer for more comprehensive coverage. Deoxynucleotide triphosphates (dNTPs) – dATP, dCTP, dGTP, and dTTP – are also essential for this reaction.

3. Removal of mRNA Template

After first-strand cDNA synthesis, the mRNA template is removed. This is commonly achieved by treating the reaction mixture with RNase H, an enzyme that specifically degrades RNA in RNA-DNA hybrids. Alternatively, alkaline hydrolysis can be used, though it requires careful optimization to avoid damaging the newly synthesized cDNA.

4. Second Strand cDNA Synthesis

The second strand of cDNA is synthesized using DNA polymerase I. This enzyme utilizes the single-stranded cDNA as a template and requires a primer. Several methods can be employed for priming:

• Nick Translation: DNA polymerase I creates nicks in the cDNA strand, and then uses these nicks as primers to initiate second-strand synthesis.
• Self-priming: The 3’ end of the first strand cDNA can sometimes fold back on itself to act as a primer.
• Random Primers: Random hexamer primers can also be used.

5. End Repair and Adaptor Ligation

The resulting double-stranded cDNA often has blunt ends or recessed 3’ ends. These ends are repaired using enzymes like T4 DNA polymerase to create blunt ends. Then, short, double-stranded DNA fragments called adaptors are ligated to the blunt ends of the cDNA using DNA ligase. These adaptors contain restriction enzyme recognition sites, which are crucial for subsequent cloning steps.

6. Restriction Enzyme Digestion

The cDNA molecules, now flanked by adaptors containing restriction enzyme sites, are digested with the corresponding restriction enzyme. This creates compatible sticky ends on both the cDNA insert and the cloning vector.

7. Vector Preparation and Ligation

A suitable cloning vector, such as a plasmid, bacteriophage lambda, or a cosmid, is chosen. The vector is also digested with the same restriction enzyme used for the cDNA. This ensures that the cDNA insert and the vector have complementary sticky ends. The digested vector and cDNA insert are then mixed together in the presence of DNA ligase, which catalyzes the formation of phosphodiester bonds, joining the cDNA insert into the vector, creating a recombinant DNA molecule.

8. Transformation and Screening

The recombinant DNA molecule is introduced into host cells (typically E. coli) through a process called transformation. The transformed cells are then plated on selective media to identify those containing the recombinant plasmid. Screening methods, such as blue-white screening or colony PCR, are used to confirm the presence of the cDNA insert in the vector.