• 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.

• Complementary DNA (cDNA) cloning is termed for the gene cloning (cloning of DNA fragments) obtained from cDNA.
• The principle of cDNA cloning is that it involves the copying of mRNA transcripts into DNA, which are then inserted into bacterial plasmids and then placed into bacteria by transformation.
• At this stage, it should be clear that mRNA used for cDNA preparation is a processed transcript and not the original one transcribed from DNA.
• In-order to clone a DNA sequence that codes for a required gene product, the gene should be removed from the organism and cloned it in the vector molecule.
• A gene library is a random collection of cloned fragments in an appropriate vector that particularly consists of all the genetic information about that species.
• There are two methods for the formation of gene libraries. They are:
  • Complementary DNA (cDNA)
  • Genomic DNA libraries

Steps of cDNA cloning:

1. Isolation of mRNA
2. Synthesis of first strand of cDNA
3. Synthesis of second strand of cDNA
4. Cloning of cDNA
5. Introduction to host cells
6. Clone selection

1. Isolation of mRNA:

• A crude extract of the tissue with the gene of interest is prepared.
• The extract must be free from proteins, polysaccharides and all other contaminants.
• The technique of oligo-deoxythymine (oligo-dT) cellulose chromatography is used for the further purification of many eukaryotic mRNAs from the total or polysomal fraction.
• mRNAs consist of poly A (adenosine residues) tail at their 3′ end.
• Under favourable conditions, this tail will bind to a string of thymidine residues immobilized on cellulose and then poly (A)⁺ fraction can be eluted.
• Two or three passages of the poly (A)⁺ fraction through such a column produces a fraction highly enriched for mRNA.
• This fraction includes different mRNA sequences, however certain techniques can be employed for extracting a particular mRNA species.
• After the preparation of the fraction, it is essential to confirm if the extracted mRNA consists of the sequence of interest.
• It is performed by translation of mRNA in vitro and identification of suitable polypeptides in the products obtained.

2. Synthesis of first strand of cDNA:

• Reverse transcriptase is a RNA dependent DNA polymerase which is used to copy the mRNA fraction into the first strand of DNA.
• This enzyme, like all other DNA polymerases, can only add residues at the 3′-OH group of an existing primer, which is base paired with the template.
• The most commonly used primer is oligo-dT for cloning of cDNAs.
• Oligo-dT primer is 12-18 nucleotides in length, that binds to the poly (A) tract at the 3′ end of mRNA molecules.
• The RNA strand of the resulting RNA-DNA hybrid is destroyed prior to second strand synthesis through alkaline hydrolysis.

3. Synthesis of second strand of cDNA:

• The second strand of cDNA can be synthesized by two techniques. They are:
• i. Self-priming cDNA:
  • In Self-priming, the mRNA hybrid obtained is denaturated for the synthesis of  second strand on the single strand of cDNA by the klenow fragment of DNA polymerase I.
  • The transitory hairpin structure at the 3′ end of single-stranded DNA can be used to prime the synthesis of second strand of cDNA by the klenow fragment of Escherichia coli DNA polymerase I.
  • Single-strand specific S1 nuclease digests the hairpin loop and any single-stranded overhung at the other end.
  • The ultimate product is a population of double-stranded, blunt-ended DNA molecules complementray to the original mRNA fraction.
• ii. Replacement synthesis:
  • In this method, the cDNA:mRNA hybrid works as a template for a nick translation reaction.
  • In the mRNA strand of the hybrid, RNase H produces nicks and gaps, creating a series of RNA primers.
  • These RNA primers are used by E. coli DNA polymerase I during the synthesis of second strand of cDNA.
  • The advantages of this technique are:
    • –  very efficient
    • –  can be performed directly using the products of the first strand reaction
    • – eliminates the need to use nuclease S1 to cleave the single-stranded hairpin loop in the double stranded cDNA.

4. Cloning of cDNA:

• The most frequently used technique for cloning cDNAs involves the addition of complementary homopolymeric tracts to double stranded cDNA and to the plasmid vector.
• To the cDNA, strings of cytosine residues are added using the enzyme terminal transferase to form oligo-dC tails on the 3′ ends.
• Likewise, a plasmid is cut open at a unique restriction endonuclease site and tailed with oligo-dG.
• Now, the vector and the double stranded cDNA are joined by hydrogen bonding between the complementary homopolymers.
• It results in the formation of open circular hybrid molecules capable of transforming E. coli.

5. Introduction to host cells:

• For the transforming of bacteria, the recombinant plasmids are used, usually the E. coli K-12 strain.
• The uptake of plasmid molecules from the surrounding medium is performed by E. coli cells treated with calcium chloride.
• Any gaps in the recombinant plasmid will be repaired by the host cells.
• The transformed bacteria can be isolated from non-transformed ones on the basis of antibiotic resistance.
• Majority of cloning plasmids contain two antibiotic resistance genes, one of which is destroyed during cloning.
• For instance, in the case of pBR322, cloning into unique PstI site destroys ampicillin resistance but leaves tetracycline resistance intact.
• Bacteria transformed with a recombinant plasmid will be sensitive to ampicillin but resistant to tetracycline.

6. Clone selection:

• The antibiotic resistance selection already performed has recognized which clones carry a recombinant plasmid, however there will be thousands of various inserts.
• The cloning process generally commences with a whole population of mRNA sequences.
• Selection of clones carrying the sequence of interest is the tough job.
• If the gene is expressed, then the simplest selection is to screen for the presence of the protein.
• It can be screened either by bacterial phenotype it produces or by the protein detection methods usually based on immunological or enzymological techniques.
• If the protein is not expressed, then other methods such as nucleic acid hybridization are used.
• Identification of the gene is discussed after the genomic DNA cloning.

cDNA cloning: Principle and steps involved in cDNA cloning

The post cDNA cloning: Principle and steps involved in cDNA cloning appeared first on Online Biology Notes.