cDNA library: Process of cDNA library, Advantages, and Disadvantages

cDNA library:

cDNA library: Process of construction of cDNA library, Advantages, and Disadvantages

cDNA library:

A cDNA is a DNA copy that is generated from messenger RNA (mRNA) with the help of the enzyme reverse transcriptase.

A set of cDNA fragments, each of which has been cloned into a separate vector molecule constituting part of the organism’s transcriptome and stored as a library, is known as a cDNA library.

Principle of cDNA library:

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

The process involved in the construction of the cDNA library:

  • First, the mRNA is collected and purified from the remaining RNAs.
  • Many other methods for purifying RNA are available, such as triazole extraction and column purification.
  • By using oligomeric DT-nucleotide-coated resins, a column purification is performed in which only the mRNA with the poly-A tail can bind.
  • By using oligomeric dT-nucleotide-coated resins, a column purification is performed in which only the mRNA with the poly-A tail can bind.
  • The remaining RNAs are eluted.
  • The mRNA is eluted using an elution buffer and also some heat to separate the mRNA strands from oligo-dT.

cDNA construction:

There are several different methods for constructing cDNAs. These will be discussed as follows:

i). The RNAse method:

Principle:

  • By the use of reverse transcriptase, 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 an RNA: DNA duplex.

Step II: Replacing RNA strand with DNA strand:

  • The RNA strand is replaced by the DNA strand with the help of the 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 is 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′ ends 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 are used.
  • The consequence of this is that at its3′ end, the DNA strand now has a known sequence. DCTP or dATP is 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 which 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 reverse transcriptase and a primer from the coding sequence.
  • It removes the unincorporated primer and tail 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 relevant 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 separate

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 lead 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 the cDNA library:

  • There are two main advantages of a cDNA library.
  • First, it is enriched with fragments of actively transcribed genes.
  • Second, introns do not destroy the cloned sequences; When the goal is to produce a eukaryotic protein in bacteria, introns pose a problem because most bacteria have no way of eliminating the introns.

Disadvantages of the cDNA library:

  • A cDNA library has the disadvantage that it only contains sequences that are present in mature mRNA.
  • There are no introns or 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 cDNA library.
  • It is also important to remember that only certain gene sequences that are expressed in the tissue from which the RNA was isolated make up the cDNA library.
  • Furthermore, in a cDNA library, the frequency of a particular DNA sequence depends on the frequency of the corresponding mRNA in the given tissue.
  • In contrast, in a genomic DNA library, almost all genes are present with the same frequency.

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cDNA library: Process of construction of cDNA library, Advantages, and Disadvantages

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