Notes on cDNA Library | DNA Libraries

In this article we will discuss about cDNA Library:- 1. Meaning of cDNA Library 2. Principle of cDNA Library 3. Vectors used in the Construction 4. Procedure in the Construction 5. Advantages 6. Disadvantages 7. Applications.

Meaning of cDNA Library:

A cDNA library is defined as a collection of cDNA fragments, each of which has been cloned into a separate vector molecule.

Principle of cDNA Library:

In the case of cDNA libraries we produce DNA copies of the RNA sequences (usually the mRNA) of an organism and clone them. It is called a cDNA library because all the DNA in this library is complementary to mRNA and are produced by the reverse transcription of the latter.

Much of eukaryotic DNA consists of repetitive sequences that are not transcribed into mRNA and the sequences are not repre­sented in a cDNA library. It must be noted that prokaryotes and lower eukaryotes do not con­tain introns, and preparation of cDNA is generally unnecessary for these organisms. Hence, cDNA libraries are produced only from higher eukaryotes.

Vectors used in the Construction of cDNA Library:

Both the bacterial and bacteriophage DNA are used as vectors in the construction of cDNA library.

The following table give a detailed in­formation:

Procedure in the Construction of cDNA Library:

The steps involved in the construction of a cDNA library are as follows:

1. Extraction of mRNA from the eukaryotic Cell:

Firstly, the mRNA is obtained and purified from the rest of the RNAs. Several methods exist for purifying RNA such as trizol extrac­tion and column purification. Column puri­fication is done by using oligomeric dT nucle­otide coated resins where only the mRNA hav­ing the poly-A tail will bind.

The rest of the RNAs are eluted out. The mRNA is eluted by using eluting buffer and some heat to sepa­rate the mRNA strands from oligo-dT.

2. Construction of cDNA from the Ex­tracted mRNA (Fig. 6.4):

There are different strategies for the construc­tion of a cDNA. These are discussed as follows:

(a) The RNase Method:

The principle of this method is that a complementary DNA strand is synthesized using reverse tran­scriptase to make an RNA: DNA duplex. The RNA strand is then nicked and replaced by DNA. In this method the first step is to anneal a chemically synthesized oligo-dT primer to the 3′ polyA-tail of the RNA.

The primer is typically 10-15 resi­dues long, and it primes (by providing a free 3′ end) the synthesis of the first DNA strand in the presence of reverse trans­criptase and deoxyribonucleotides. This leaves an RNA: DNA duplex.

The next step is to replace the RNA strand with a DNA strand. This is done by using RNase H enzyme which removes the RNA from RNA: DNA duplex. The DNA strand thus left behind is then considered as the template and the second DNA strand is synthesized by the action of DNA poly­merase II.

(b) The Self-Priming method:

This involved the use of an oligo-dT primer annealing at the polyadenylate tail of the mRNA to prime first DNA strand synthesis against the mRNA. This cDNA thus formed has the tendency to transiently fold back on itself, forming a hairpin loop. This results in the self-priming of the second strand.

After the synthesis of the second DNA strand, this loop must be cleaved with a single-strand-specific nuclease, e.g., SI nuclease, to allow insertion into the clon­ing vector. This method has a serious disadvantage. The cleavage with SI nuclease results in the loss of a certain amount of sequence at the 5′ end of the clone.

(c) Land et al. Strategy:

After first-strand synthesis, which is primed with an oligo- dT primer as usual, the cDNA is tailed with a string of cytidine residues using the en­zyme terminal transferase. This artificial oligo-dC tail is then used as an annealing site for a synthetic oligo-dG primer, allow­ing synthesis of the second strand.

(d) Homopolymer Tailing:

This approach uses the enzyme terminal transferase, which can polymerize nucleotides onto the 3′-hydroxyl of both DNA and RNA mol­ecules. We carry out the synthesis of the first DNA strand essentially as before, to produce an RNA: DNA hybrid.

We then use terminal transferase and a single deoxyribonucleotide to add tails of that nucleotide to the 3′ ends of both RNA and DNA strands. The result of this is that the DNA strand now has a known sequence at its 3′ end Typically, dCTP or dATP are used.

A complementary oligomer (synthesized chemically) can now be annealed and used as a primer to direct second strand syn­thesis. This oligomer (and also the one used for first strand synthesis) may addi­tionally incorporate a restriction site, to help in cloning the resulting double- stranded cDNA.

(e) Rapid Amplification of cDNA Ends (RACE):

It is sometimes the case that we wish to clone a particular cDNA for which we already have some sequence data, but with particular emphasis on the integrity of the 5′ or 3′ ends. RACE techniques (Rapid Amplification of cDNA Ends) are available for this. The RACE methods are divided into 3’RACE and 5’RACE, accord­ing to which end of the cDNA we are in­terested in.

(a) 3’RACE:

In this type of RACE, reverse transcriptase synthesis of a first DNA strand is carried out using a modified oligo-dT primer. This primer comprises a stretch of unique adaptor se­quence followed by an oligo-dT stretch. The first strand synthesis is followed by a second strand synthesis using a primer internal to the coding sequence of interest.

This is followed by PCR using

(i) The same internal primer and ‘

(ii) The adaptor sequence (i.e., omitting the oligo-dT). Although in theory it should be possible to use a simple oligo- dT primer throughout instead of the adaptor-oligo-dT and adaptor combi­nation, the low melting temperature for an oligo-dT primer may interfere with the subsequent rounds of PCR.

(b) 5’RACE:

In this type of RACE first cDNA strand is synthesized with re- verse transcriptase and a primer from within the coding sequence. Unincor­porated primer is removed and the cDNA strands are tailed with oligo-dA. A second cDNA strand is then synthe­sized with an adaptor-oligo-dT primer.

The resulting double-stranded mol­ecules are then subject to PCR using

(i) A primer nested within the coding region and

(ii) The adaptor sequence. A nested primer is used in the final PCR to improve specificity. The adap­tor sequence is used in the PCR be­cause of the low melting temperature of a simple oligo-dT primer, as in 3’RACE above. A number of kits for RACE are commercially available.

3. Cloning the c-DNA:

(a) Linkers:

The RNaseH and homopolymer tailing methods ultimately generate a col­lection of double-stranded, blunt-ended cDNA molecules. They must now be at­tached to the vector molecules. This could be done by blunt-ended ligation, or by the addition of linkers, digestion with the rel­evant enzyme and ligation into vector.

(b) Incorporation of Restriction Sites:

It is possible to adapt the homopolymer tailing method by using primers that are modi­fied to incorporate restriction. In the dia­gram shown next page, the oligo-dT primer is modified to contain a restriction site (in the figure, a Sail site GTCGAC).

The 3′ end of the newly synthesized first cDNA strand is tailed with C’s. An oligo-dG primer, again preceded by a Sail site within a short double-stranded region of the oligonucleotide, is then used for sec­ond-strand synthesis.

Note that this method requires the use of an oligonucleo­tide containing a double-stranded region. Such oligonucleotides are made by synthe­sizing the two strands separately and then allowing them to anneal to one another.

(c) Homopolymer Tailing of cDNA:

An­other option is to use terminal transferase again. Treatment of the blunt-ended double-stranded cDNA with terminal transferase and dCTP leads to the poly­merization of several C residues (typically 20 or so) to the 3′ hydroxyl at each end.

Treatment of the vector with terminal transferase and dGTP leads to the incor­poration of several G residues onto the ends of the vector. (Alternatively, dATP and dTTP can be used.) The vector and cDNA can now anneal, and the base-paired region is often so extensive that treatment with DNA ligase is unnecessary.

In fact, there may be gaps rather than nicks at the vector insert boundaries, but these are re­paired by physiological processes once the recombinant molecules have been intro­duced into a host.

Advantages of cDNA Library:

A cDNA library has two additional advantages. First, it is enriched with fragments from ac­tively transcribed genes. Second, introns do not interrupt the cloned sequences; introns would pose a problem when the goal is to pro­duce a eukaryotic protein in bacteria, because most bacteria have no means of removing the introns.

Disadvantages of cDNA Library:

The disadvantage of a cDNA library is that it contains only sequences that are present in mature mRNA. Introns and any other se­quences that are altered after transcription are not present; sequences, such as promoters and enhancers, that are not transcribed into RNA also are not present in a cDNA library.

It is also important to note that the cDNA library represents only those gene sequences ex­pressed in the tissue from which the RNA was isolated. Furthermore, the frequency of a par­ticular DNA sequence in a cDNA library depends on the abundance of the corresponding mRNA in the given tissue. In contrast, almost all genes are present at the same frequency in a genomic DNA library.

Applications of cDNA Library:

Following are the applications of cDNA librar­ies:

1. Discovery of novel genes.

2. Cloning of full-length cDNA molecules for in vitro study of gene function.

3. Study of the repertoire of mRNAs ex­pressed in different cells or tissues.

4. Study of alternative splicing in differ­ent cells or tissues.