Making of a cDNA library - Discover Biotech

In this article, I briefly describe the making of a cDNA library, which starts with the synthesis of cDNA.

Table of Contents

Gene library

The collection of different DNA sequences from an organism, which is the total genomic DNA of the organism, makes a gene library. Each DNA sequence is cloned into a vector for ease of purification, storage, and analysis. The first step in the construction of a gene library is to extract the organism’s DNA from cells. After the DNA is extracted, it is followed by digestion with a restriction enzyme to cut it into fragments of a specific size. Then, these fragments are inserted into a vector by the enzyme DNA ligase. For easy amplification and analysis of specific clones from the library, the vector is taken up by a host organism like a population of E.coli or yeast.

Depending upon the source of DNA, two types of gene libraries can be prepared. If the DNA is genomic, the library is called a genomic library. If the DNA is a copy of an mRNA population, which is cDNA, then the library is a cDNA library. A library is called representative when the starting material is well represented by it, and the library contains all the original sequences.

cDNA library

A collection of cloned DNA sequences complementary to the mRNA, extracted from an organism or tissue, constitutes a cDNA library. cDNA is produced from fully transcribed mRNA found in the nucleus. Therefore, it contains only the expressed genes of an organism. The cDNAs that are made from eukaryotic mRNAs have no intron sequences. Thus, they can be used to express the encoded protein in E.coli. These cDNA libraries are thus very useful.

In eukaryotes, the 3′ tails of polyadenylated mRNAs provide a useful method for isolating eukaryotic mRNAs. A poly -(A) tail (consists of long sequences of adenine nucleotides) separates mRNA from rRNA and tRNA. Thus, it can be used as a primer site for reverse transcription. Oligo(dT) can be bound to the poly(A) tail and used to recover the mRNA. The mRNA preparation for cDNA cloning is used by either translating the mRNA or analyzing it by gel electrophoresis. It is useful to fractionate or enrich the mRNA before cDNA cloning. Fractionation is usually performed based on size. mRNAs of different sizes are recovered from agarose gels. Enrichment is usually carried out by hybridization.

Synthesis of cDNA

The enzyme reverse transcriptase is an RNA-dependent DNA polymerase used for the synthesis of cDNA. It is isolated from a retrovirus (generally obtained from Avian Mycoblastosis Virus or Moloney Murine Leukemia Virus ). The enzyme synthesizes a DNA polynucleotide strand complementary to an existing mRNA strand. While making a copy of the mRNA template, the enzyme needs a primer.

Eukaryotic mRNAs possess a poly-A tail at their 3′-ends. Therefore, a poly-T oligonucleotide is used as a primer. However, in other RNAs lacking a poly-A tail, e.g., prokaryotic mRNA, rRNA, and RNA virus genomes, a poly-A tail is added to the 3′-end of the RNA. This makes the RNA analogous to the eukaryotic mRNA. All four dNTPs (dTTP,dGTP,dCTP,dATP) must be added. The primer gets annealed to the 3′-end of the mRNA. The reverse transcriptase helps to extend the 3′-end of the primer using the mRNA strand as a template. This is known as the “first strand reaction”. This results in the production of RNA-DNA hybrid molecules. To get the single-stranded cDNA, the RNA strand of this RNA-DNA hybrid is digested by using RNase H or alkaline hydrolysis.

Formation of a hairpin loop structure

The resulting single-stranded cDNA (ss cDNA) is used as the template to produce the second DNA strand. During the synthesis of reverse transcriptase, a short complementary tail at the 5′ end of the cDNA is produced. Thus, this acts as a double-stranded primer sequence for other polymerases. The short tail loops back onto the ss cDNA template ( the hairpin loop ) and provides the primer for the polymerase to start the synthesis of the new DNA strand, producing a double-stranded cDNA (ds cDNA). The complementary strand of the cDNA single strand is synthesized either by the reverse transcriptase itself or by the Klenow fragment of E. coli DNA polymerase I. A single strand-specific nuclease cleaves the hairpin loop to yield a regular DNA duplex (figure 1).

**Figure 1: Synthesis of double-stranded cDNA by the self-priming method**

A second method of making cDNA

To make a full-length cDNA, the 3′ end of the mRNA strand is tailed, and a complementary primer is used for making the second strand. The enzyme reverse transcriptase displays terminal transferase activity on reaching the end of the RNA template. Terminal transferase adds nucleotides to the 3′ end of single or double-stranded nucleic acids without the requirement of a template. With one dNTP, for example, dCTP, it will add a homopolymeric tail of C residues to the 3′ ends of the mRNA-cDNA duplex.

The mRNA strand is destroyed with alkali. After this, oligo(dG) can be used as a primer for the synthesis of the second strand using either reverse transcriptase or the Klenow fragment of E.coli DNA polymerase I (figure 2). The degradation of the newly synthesized cDNAs is avoided by the Klenow polymerase. The 3′-end of the first strand may protrude beyond the 5′ end of the second strand depending on the relative lengths of the poly(dC) tail and the oligo(dG) primer. A single strand-specific nuclease removes the protruding ends to get a regular duplex of cDNA.

**Figure 2: Synthesis of cDNA by primer addition**

cDNA ends undergo treatment

The blunt ends of the duplex cDNA are manipulated before cloning into the vector. Blunt end ligation of large fragments is not efficient. So, special nucleic acid linkers are added to create sticky ends for cloning. The regular duplex of cDNA is treated with the Klenow fragment of DNA polymerase I and dNTPs to fill in any missing 3′ nucleotides. If the linkers contain the same restriction enzyme site that is likely to be present in the cDNA(such as EcoRI), then the cDNA should undergo methylation using EcoRI methylase before the addition of the linkers. Thus, the restriction enzyme can’t cleave the cDNA internally. Then, with the help of T4 DNA ligase, the linkers can be ligated to the blunt-ended, duplex cDNA. This step will add one linker to each end of 5′-phosphorylated cDNA if the linkers are not phosphorylated.

The last step includes digestion with the restriction enzyme EcoRI, which generates sticky ends ready for ligation to the vector (figure 3). Adapter molecules have preformed ‘sticky’ ends and can be used as an alternative way for preparing the cDNA ends for cloning. This avoids the methylation of the cDNA.

**Figure 3: Treatment of cDNA ends by addition of a linker**

Vector ligation

λ phage vectors are generally used for the expression of cDNA libraries. The enzyme alkaline phosphatase dephosphorylates the vector, and this prevents the rejoining of the vector fragments during ligation. Thus, by the joining of vector and cDNA, only recombinant molecules are produced. The vector λgt11 has an EcoRI site placed near the C terminus of itslacZ gene, enabling expression of the cDNA as part of a large β-galactosidase fusion protein. T4 DNA ligase helps to ligate the vector to cDNA, and the recombinant molecules are either packaged or transformed to create the cDNA library.

Two specific primers introduce very rare restriction recognition sites into the duplex cDNA. These sites are present in the plasmid vector and permit directional cloning. The cDNA synthesis procedure relies on reverse transcriptase acting as a terminal transferase and adding C residues to the 3′ end of the first strand. When the specific sense primer containing oligo d(G) is added, the reverse transcriptase makes the second strand.

Conclusion

A collection of cloned DNA sequences complementary to the mRNA, extracted from an organism or tissue, constitutes a cDNA library. cDNA is produced from fully transcribed mRNA found in the nucleus. So, it contains only the expressed genes of an organism. The enzyme reverse transcriptase helps in the synthesis of cDNA.

Synthesis of cDNA can be done either by the self-priming method or by the addition of a primer. Treatment of blunt-ended cDNAs is done by the addition of special nucleic acid linkers. The addition of linkers creates sticky ends for cloning. With the help of T4 DNA ligase, the linkers can be ligated to the blunt-ended, duplex cDNA.