• A copy of DNA generated from messenger RNA (mRNA) with the help of enzyme reverse transcriptase is termed as cDNA.
• A set of cDNA fragments, each of which has been cloned into a separate vector molecule, which constitute a some portion of transcriptome of the organism and stored as a library is known as a cDNA library.

Principle of cDNA library:

• To construct cDNA libraries, DNA copies from mRNA sequences of organism are produced and then they are cloned.
• The term cDNA is given as all the DNA in the library are complementary to the mRNAs and are produced by reverse transcription of mRNAs.
• Most eukaryotic DNA consists of repeated sequences that are not transcribed into mRNA, and in a cDNA library the sequences are not represented.
• It should be remembered that prokaryotes and lower eukaryotes do not contain introns, and cDNA preparation for these species is usually needless.
• Therefore, cDNA libraries are only created from higher eukaryotes.
• For the construction of cDNA library, both the bacterial and bacteriophage DNA can be used as vectors.

Process involved in the construction of cDNA library:

1. Extraction of mRNA from the eukaryotic cell:

• Firstly, the mRNA from the remaining RNAs is collected and purified.
• Many other methods are available for purifying RNA like trizol extrac­tion and column purification.
• By using oligomeric dT nucleotide coated resins, column purification is performed where only the mRNA that has the poly-A tail can bind.
• By using oligomeric dT nucleotide coated resins, column purification is performed where only the mRNA that has the poly-A tail can bind.
• The remaining RNAs are eluted.
• The mRNA is eluted using eluting buffer and also some heat to sepa­rate the mRNA strands from oligo-dT.

2. cDNA construction:

• For the construction of cDNAs, there are several different methods. These are discussed as follows:

i). The RNAse method:

• Principle:
  • By the use of reverse transcriptase, a complementary DNA is synthesized to form an RNA:DNA duplex. Now, the RNA strand is nicked and then replaced by DNA.
• Steps:
• Step I: Annealing:
  • A chemically synthesized oligo-dT primer is annealed to the 3’ polyA-tail of the RNA. The primer is usually 10-15 residues long.
  • In the presence of reverse transcriptase and deoxyribonucleotides, it primes the synthesis of the first DNA strand. This leaves a RNA:DNA duplex.
• Step II: Replacing RNA strand with DNA strand:
  • The RNA strand is replaced by DNA strand by the help of enzyme RNAse H.
  • RNase enzyme removes the RNA from RNA:DNA duplex. The DNA strand which is left behind now acts as a template and the other DNA strand synthesized by the DNA polymerase II.

ii). The self-priming method:

• In this method, the oligo-dT primer is annealed at the polyadenylate tail of the mRNA to prime the first DNA strand synthesis against the mRNA.
• This cDNA, thus formed, tends to fold back on itself temporarily, creating a hairpin loop.
• This results in the second strand’s self-priming.
• This loop must be cleaved with a single-strand-specific nuclease, e.g., SI nuclease, after the synthesis of the second DNA strand to allow insertion into the cloning vector.
• There is a serious drawback to this method.
• At the 5′ end of the clone, cleavage with SI nuclease results in the loss of a certain amount of sequence.

iii). Land et al. strategy:

• The cDNA is tailed with a string of cytidine residues using the enzyme terminal transferase following first-strand synthesis, which is primed with an oligo-dT primer as usual.
• For a synthetic oligo-dG primer, this artificial oligo-dC tail is then used as an annealing site, allowing the second strand to be synthesized.

iv).  Homopolymer tailing:

• The enzyme terminal transferase that can polymerize nucleotides into the 3′-hydroxyl of both DNA and RNA molecules is used in this method.
• In order to generate an RNA: DNA hybrid, the synthesis of the first DNA strand is performed as before.
• In order to add nucleotide tails to the3′ ends of both RNA and DNA strands, then  terminal transferase and a single deoxyribonucleotide is used.
• The consequence of this is that at its3′ end, the DNA strand now has a known sequence. DCTP or dATP are usually used.
• A complementary oligomer (chemically synthesized) can now be annealed and used as a primer to direct the synthesis of the second strand.
• To assist in cloning the resulting double-stranded cDNA, this oligomer (and also the one used for first strand synthesis) can additionally incorporate a restriction site.

v). Rapid amplification of cDNA ends:

• The RACE techniques are split into 3’RACE and 5’RACE, according to the end of the cDNA in which we are interested.
• a.  3’ RACE:
  • Reverse transcriptase synthesis of a first DNA strand is performed using a modified oligo-dT primer in this type of RACE.
  • This primer involves an extension of a particular adaptor sequence followed by an oligo-dT stretch.
  • The first strand synthesis is followed by a second strand synthesis that used a primer internal to the coding sequence of interest.
  • This is accompanied by PCR that uses
    • i. The same internal primer.
    • ii. Sequence of the adaptor (i.e., omitting the oligo-dT). Although it should be possible to use a simple oligo-dT primer in theory instead of the adaptor-oligo-dT and adaptor combination, the low melting temperature can interfere with the subsequent PCR rounds for an oligo-dT primer.
• b. 5’ RACE:
  • The first cDNA strand of this type of RACE is synthesized with re-verse transcriptase and a primer from the coding sequence.
  • It removes the unincorporated primer and tails the cDNA strands with oligo-dA.
  • With an adaptor-oligo-dT primer, a second cDNA strand is then synthesized.
  • The double-stranded molecules resulting from this are then subjected to PCR using
    • i.  A primer nested within the coding region and
    • ii. In the final PCR, a nested primer is used to maximize specificity. Due to the low melting temperature of a basic oligo-dT primer, the adaptor sequence is used in the PCR, as in the 3’RACE above. A variety of kits are commercially available for RACE.

3. cDNA cloning:

a. Linkers:

• In the end, the methods of RNaseH and homopolymer tailing generate a collection of double-stranded, blunt-ended cDNA molecules.
• The vector molecules must now be bound to them.
• This could be achieved by blunt-ended ligation, digestion with the rela-evant enzyme and ligation into the vector, or by adding linkers.

b. Incorporation of restriction sites:

• The homopolymer tailing technique can be adapted by using primers that are adjusted to incorporate restrictions.
• The 3 ‘end of the first cDNA strand, recently synthesized, is tailed with C’s.
• An oligo-dG primer, again preceded by a sail site within the oligonucleotide’s short double-stranded region, is then used for second-strand synthesis.
• The use of an oligonucleotide containing a double-stranded region is necessary in this process.
• Such oligonucleotides are formed by separately synthesizing the two strands and then allowing them to anneal with each other.

c. Homopolymer Tailing of cDNA:

• Another idea is to re-use terminal transferase.
• Treatment with terminal transferase and dCTP of blunt-ended double-stranded cDNA leads to the polymerization of several C residues (typically 20 or so) to 3′ hydroxyl at each end.
• The terminal transferase and dGTP treatment of the vector leads to the inclusion of several G residues on the ends of the vector. It is possible to use dATP and dTTP alternatively.
• It is now possible to anneal the vector and cDNA, and the base-paired region is often so extensive that DNA ligase treatment is unnecessary.
• There may actually be gaps rather than nicks at the edges of the vector insert, but once the recombinant molecules have been inserted into a host, these are repaired by physiological processes.

Advantages of cDNA library:

• There are two major benefits of a cDNA library.
• First, it is enriched with fragments from genes that have been actively transcribed.
• Second, introns do not disrupt the cloned sequences; if the goal is to create a eukaryotic protein in bacteria, introns will pose a problem, since most bacteria have no means of eliminating the introns.

Disadvantages of cDNA library:

• A cDNA library has the drawback that it only includes sequences that are present in mature mRNA.
• There are no introns and any other sequences that are modified during transcription; sequences that are not transcribed into RNA, such as promoters and enhancers, are also not present in a library of cDNA.
• It is also important to remember that only certain gene sequences expressed in the tissue from which the RNA has been isolated constitute the cDNA library.
• In addition, in a cDNA library, the frequency of a specific DNA sequence depends on the abundance of the corresponding mRNA in the given tissue.
• In contrast, in a genomic DNA library, almost all genes are present at the same frequency.

The post cDNA library: Process of construction of cDNA library, Advantages and Disadvantages appeared first on Online Biology Notes.