15.2: Make and Screen a cDNA Library - Biology

15.2: Make and Screen a cDNA Library - Biology

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The first step in making a cDNA library is to isolate cellular mRNA. This mRNA extract should represent all of the transcripts in the cells at the time of isolation, or the cell’s transcriptome. This term is used by analogy to genome. However, a genome is all of the genetic information of an organism. In contrast, a transcriptome (usually eukaryotic) reflects all of the genes expressed in a given cell type at a moment in time. Reversetranscribed cDNAs from an mRNA extract are also referred to as a transcriptome…, and likewise, a cDNA library. A cDNA library is a tube full of bacterial cells that have taken up (i.e., been transformed with) plasmids recombined with cDNAs. cDNA libraries made from mRNAs taken from different cell types or the same cells grown under different conditions are in effect, different transcriptomes. Each reflects mRNAs transcribed in cells at the moment of their extraction. When cells in a cDNA library are spread out on a nutrient agar petri dish, each cell grows into a colony of cells; each cell in the colony is a clone of a starting cell. cDNA libraries can be used isolate and sequence the DNA encoding a polypeptide that you are studying.

Recall that the mature mRNA in eukaryotic cells has been spliced. This means that cDNAs from eukaryotic cells do not include introns. Introns, as well as sequences of enhancers and other regulatory elements in and surrounding a gene must be studied in genomic libraries, to be discussed later. Here we look at how to make a cDNA library.

A. cDNA Construction

mRNA is only a few percent of a eukaryotic cell; most is rRNA. But that small amount of mRNA can be separated from other cellular RNAs by virtue of their 3’ poly(A) tails. Simply pass a total RNA extract over an oligo-d(T) column (illustrated below).

The strings of thymidine (T) can H-bond with the poly(A) tails of mRNAs, tethering them to the column. All RNAs without a 3’ poly(A) tail will flow through the column as waste. A second buffer is passed over the column to destabilize the A-T H-bonds to allow elution of an mRNA fraction. When free’ oligo d(T) is added to the eluted mRNA, it forms H-bonds with the poly(A) tails of the mRNAs, serving as a primer for the synthesis of cDNA copies of the poly(A) mRNAs originally in the cells. Finally, four deoxynucleotide DNA precursors and reverse transcriptase (originally isolated from chicken retrovirus-infected cells) are added to start reverse transcription. The synthesis of a cDNA strand complementary to an mRNA is shown below.

After heating to separate the cDNAs from the mRNAs, the cDNA is replicated to produce double-stranded, or (ds)cDNA, as illustrated below.

Synthesis of the second cDNA strand is also catalyzed by reverse transcriptase! The enzyme recognizes DNA as well as RNA templates, and has the same 5’-to-3’ DNA polymerizing activity as DNA polymerases. After 2nd cDNA strand synthesis, S1 nuclease (a single-stranded endonuclease originally isolated from an East Asian fungus!) is added to open the loop of the (ds) cDNA structure and trim the rest of the single-stranded DNA. What remains is the (ds) cDNA.

B. Cloning cDNAs into Plasmid Vectors

To understand cDNA cloning and other aspects of making recombinant DNA, we need to talk a bit more about the recombinant DNA tool kit. In addition to reverse transcriptase and S1 nuclease, other necessary enzymes in the ‘kit’ include restriction endonucleases (restriction enzymes) and DNA ligase. The natural function of restriction enzymes in bacteria is to recognize specific restriction site sequences in phage DNA (most often palindromic DNA sequences), hydrolyze it and thus avoid infection.

Restriction enzymes that make a scissors cut through the two strands of the double helix leaves blunt ends. Restriction enzymes that make a staggered cut on each strand at their restriction site leave behind complementary (‘sticky’) ends (below).

If you mix two of double-stranded DNA fragments with the same sticky ends from different sources (e.g., different species), they will form H-bonds at their complementary ends, making it easy to recombine plasmid DNA with (ds)cDNA, that have the same complementary ‘sticky ends’. Using the language of recombinant DNA technologies, let’s look at how plasmid vectors and cDNAs can be made to recombine.

1. Preparing Recombinant Plasmid Vectors Containing cDNA Inserts

Vectors are carrier DNAs engineered to recombine with foreign DNAs of interest. When a recombinant vector with its foreign DNA insert gets into a host cell, it can replicate many copies of itself, enough in fact for easy isolation and study. cDNAs are typically inserted into plasmid vectors that are usually purchased “off-the-shelf”. They can be cut with a restriction enzyme at a suitable location, leaving those sticky ends. On the other hand, it would not do to digest (ds)cDNA with restriction endonucleases since the goal is not to clone cDNA fragments, but entire cDNA molecules. Therefore, it will be necessary to attach linkers to either end of the (ds)cDNAs. Plasmid DNAs and cDNA-linker constructs can then be digested with the same restriction enzyme to produce compatible ‘sticky ends’. Steps in the preparation of vector and (ds)cDNA for recombination are shown below.

To prepare for recombination, a plasmid vector is digested with a restriction enzyme to open the DNA circle. To have compatible sticky ends, double-stranded cDNAs to be inserted are mixed with linkers and DNA ligase to put a linker DNA at both ends of the (ds) cDNA. DNA ligase is another tool in the recombinant DNA toolkit. Linkers are short, synthetic double-stranded DNA oligomers containing restriction sites recognized and cut by the same restriction enzyme as the plasmid. Once the linkers are attached to the ends of the plasmid DNAs, they are digested with the appropriate restriction enzyme. This leaves both the (ds)cDNAs and the plasmid vectors with complementary sticky ends.

2. Recombining Plasmids and cDNA Inserts and Transforming Host Cells

The next step is to mix the cut plasmids with the digested linker-cDNAs in just the right proportions so that the most of the cDNA (linker) ends will anneal (form Hbonds) with the most of the sticky plasmid ends. Adding DNA ligase to the plasmid/linker-cDNA mixture forms phosphodiester bonds between plasmid and cDNA insert, completing the recombinant circle of DNA, as shown below.

In early cloning experiments, an important consideration was to generate plasmids with only one copy of a given cDNA insert, rather than lots of re-ligated plasmids with no inserts or lots of plasmids with multiple inserts. Using betterengineered vector and linker combinations, this issue became less important.

3. Transforming Host Cells with Recombinant Plasmids

The recombinant DNA molecules are now ready for ‘cloning’. They are added to E. coli (sometimes other host cells) made permeable so that they can be easily transformed. Recall that transformation as defined by Griffith is bacterial uptake of foreign DNA leading to a genetic change. The transforming principle in cloning is the recombinant plasmid! The transformation step is shown below.

The tube full of transformed cells is the cDNA Library.

After all these treatments, not all plasmid molecules in the mix are recombinant; some cells in the mix haven’t even taken up a plasmid. So when the recombinant cells are plated on agar, how do you tell which of the colonies that grow came from cells that took up a recombinant plasmid? Both the host strain of E. coli and plasmid vectors used these days were further engineered to solve this problem. One such plasmid vector carries an antibiotic resistance gene. In this case, ampicillin-sensitive cells would be transformed with recombinant plasmids containing the resistance gene. When these cells are plated on media containing ampicillin (a form of penicillin), they grow, as illustrated below.

Untransformed cells (cells that failed to take up a plasmid) lack the ampicillin resistance gene and thus, do not grow on ampicillin-medium. But, there is still a question. How can you tell whether the cells that grew were transformed by a recombinant plasmid containing a cDNA insert? It is possible that some of the transformants contain only non-recombinant plasmids that still have the ampicillin resistance gene!

To address this question, plasmids were further engineered with a streptomycin resistance gene. But this antibiotic resistance gene was also engineered to contain restriction enzyme sites in the middle of the gene. Thus, inserting a cDNA in this plasmid would disrupt and inactivate the gene. Here is how this second bit of genetic engineering enabled growth only of cells transformed with a recombinant plasmid containing a cDNA insert. We can tell transformants containing recombinant plasmids apart from those containing non-recombinant plasmids by the technique of replica plating shown (illustrated below).

After colonies grow on the ampicillin agar plate, lay a filter over the plate. The filter will pick up a few cells from each colony, in effect becoming a replica (mirror image) of the colonies on the plate. Place the replica filter on a new agar plate containing streptomycin; the new colonies that grow on the filter must be streptomycin-resistant, containing only non-recombinant plasmids. Colonies containing recombinant plasmids, those that did not grow in streptomycin are easily identified on the original ampicillin agar plate. In practice, highly efficient recombination and transformation procedures typically reveal very streptomycinresistant cells (i.e., colonies) after replica plating. In this case, ampicillin-resistant cells constitute a good cDNA library, ready for screening.

4. Identifying Colonies Containing Plasmids with Inserts of Interest

The next step is to screen the colonies from the cDNA library for those containing the specific cDNA that you’re after. This typically begins preparing multiple replica filters like the one above. Remember, these filters are replicas of bacterial cells containing recombinant plasmids that grow on ampicillin but not streptomycin. The number of replica filters that must be screened can be calculated from assumptions and formulas for estimating how many colonies must be screened to represent an entire transcriptome (i.e., the number of different mRNAs in the original cellular mRNA source). Once the requisite number of replica filters are made, they are subjected to in situ lysis to disrupt cell walls and membranes. The result is that the cell contents are released and the DNA is denatured (i.e., becomes single-stranded). The DNA then adheres to the filter in place (in situ, where the colonies were). The result of in situ lysis is a filter with faint traces of the original colony (below).

Next, a molecular probe is used to identify DNA containing the sequence of interest. The probe is often a synthetic oligonucleotide whose sequence was inferred from known amino acid sequences. These oligonucleotides are made radioactive and placed in a bag with the filter(s). DNA from cells that contained recombinant plasmids with a cDNA of interest will bind the complementary probe. The results of in situ lysis and hybridization of a radioactive probe to a replica filter are shown below.

The filters are rinsed to remove un-bound radioactive oligomer probe, and then placed on X-ray film. After a period of exposure, the film is developed. Black spots will form on the film from radioactive exposure, creating an autoradiograph of the filter. The black spots in the autoradiograph correspond to colonies on a filter that contain a recombinant plasmid with your target cDNA sequence (below).

Once a positive clone is identified on the film, the corresponding recombinant colony is located on the original plate. This colony is grown up in a liquid culture and the plasmid DNA is isolated. At that point, the cloned plasmid DNA can be sequenced and the amino acid sequence encoded by its cDNA can be inferred from the genetic code dictionary to verify that the cDNA insert in fact encodes the protein of interest. Once verified as the sequence of interest, a cloned plasmid cDNA can be made radioactive or fluorescent, and used to

  • probe for the genes from which they originated.
  • identify and quantitate the mRNA even locate the transcripts in the cells.
  • quantitatively measure amounts of specific mRNAs.

Isolated plasmid cDNAs can even be expressed in suitable cells to make the encoded protein. These days, diabetics no longer receive pig insulin, but get synthetic human insulin human made from expressed human cDNAs. Moreover, while the introduction of the polymerase chain reaction (PCR, see below) has superseded some uses of cDNAs, they still play a role in genome-level and transcriptome-level studies.

Genomic Libraries

The Future of Genomic Libraries

Genomic library construction remains an important technique in molecular biology. These resources are critical for analysis of gene function and for detection of related genes from different sources. Genomic libraries are currently in use to find novel natural products, such as antimicrobials. They are also being used to uncover and optimize new biochemical pathways, such as those needed for production of biofuels and other complex chemicals. In addition, genomic libraries remain an essential tool for assembling the vast amount of sequence information that is produced from NGS.

With the advent of synthetic biology, it is possible to manipulate fragments containing millions of base pairs, allowing the engineering of entire pathways and genomes. The current highlight of this technology is the assembly of an entire bacterial genome (Mycoplasma laboratorium) from a subset of its parental genes that were synthesized in the laboratory. Nevertheless, the available tools are still in their infancy, and the technology is expensive and time-consuming. The future of genomic libraries may lie in methods to easily construct artificial chromosomes containing any desired genetic elements by using readily accessible ‘building blocks’. Such methods may lead to completely synthetic, preprogrammed genomes, and are already in development.

A cDNA library represents a collection of only the genes that are encoded into proteins by an organism. Complementary DNA, or cDNA, is created through reverse transcription of messenger RNA, and a library of cDNAs is generated using DNA cloning technology.

The synthesis of DNA from an RNA template, via reverse transcription, produces complementary DNA ( cDNA ). Alternatively, the first-strand cDNA can be made double-stranded using DNA Polymerase I and DNA Ligase. These reaction products can be used for direct cloning without amplification.

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:

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.

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.

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.

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.

Construct high-quality full-length cDNA libraries using SuperScript reverse transcriptase and Gateway recombination cloning (no restriction enzyme digestion needed), or use standard restriction enzyme–based cloning.

  • Learn more about the SuperScript Full-Length cDNA Library Construction Kit, the commercially available kit that includes a 5′ CAP-mRNA–purification step to help ensure that your cDNA library is full-length.
  • Learn more about the CloneMiner II cDNA Library Construction Kit
  • Order cDNA Library Construction Kits


Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein– or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension ( CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ∼2 × 10 5 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

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No enrollment or registration. Freely browse and use OCW materials at your own pace. There's no signup, and no start or end dates.

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Your use of the MIT OpenCourseWare site and materials is subject to our Creative Commons License and other terms of use.

Synthetic Peptides as Antigents

Construction of a recombinant DNA library in λgt11

Genomic libraries are used for organisms such as Drosophila or yeast that have a small genomic size and few introns in their coding sequences. In this case the number of genomic recombinants that must be screened in order to isolate the gene of interest in not too large. For organisms such as mammals which have a large genome, it is necessary to use cDNA libraries. The construction of cDNA and genomic libraries has been described in detail ( Ausubel et al., 1994–1997 O’Reilly et al., 1992 Sambrook et al., 1989 ).

ShRNA Libraries

shRNA libraries permit reversible loss-of-function screening to elucidate which genes are involved in a phenotype. Each library contains multiple shRNA sequences for each target gene a true positive hit (a particular target gene) in an shRNA screen should show consistent results from the multiple shRNAs that target it. shRNA libraries may also be barcoded to allow for easy identification of the shRNA a given cell carries.

DECIPHER libraries are lentiviral barcoded shRNA libraries designed for RNAi screening. It’s important to note that these libraries are not genome-wide instead, each library is enriched for a smaller set of biologically relevant genes. The DECIPHER barcoding system makes it easy to determine which shRNAs have been enriched or depleted in a cell population each plasmid carries a unique 18 bp barcode that can be easily identified using next-generation sequencing.

As of October 2016, the DECIPHER libraries have been discontinued.

Watch the video: DNA libraries u0026 generating cDNA. Biomolecules. MCAT. Khan Academy (May 2022).