[priyasaravanan_1406]

Like a library is a place sheltering large collection of books on different subjects for public use, a DNA library involves collection of DNA sequences from various organisms, serving different purposes. These DNA sequences are stored as recombinant molecules by ligating them with suitable vectors. The two types of gene libraries are genomic library and cDNA library, categorized based on the source from where they were constructed. A genomic library holds the DNA sequences derived from genomic DNA whereas the cDNA library represents the DNA sequences generated from mRNA. The library that represents the source DNA as such can be considered as an ideal gene library.

Construction of genomic DNA library: Preparation of the genomic DNA library involves isolation of genomic DNA, purification of the genomic DNA and fragmentation of genomic DNA into desired size and then cloning of the fragmented DNA using suitable vector. The eukaryotic cell nuclei are purified by adopting digestion by protease and phenol-chloroform applied phase extraction. The derived genomic DNA is long and needs to be cut into desirable fragment sizes. Fragmentation of DNA is achieved by physical method and enzymatic method.

Physical method includes pipeting the DNA molecule or applying intensified ultrasound waves (sonication). The enzymatic method involves the use of restriction enzyme to fragment the purified DNA. The desirable DNA size suitable for the cloning vector decides the method to be used for fragmenting the DNA and the length of exposure of the DNA molecule. The distribution probability of site prone to the action of restriction enzymes limits the use of the enzymatic method which will produce shorter DNA fragments than the desired size. To overcome this problem partial digestion of the DNA molecule is done using known quantity of restriction enzyme which yields fragments of desired size. The two factors governing the selection of the restriction enzymes are the type of ends they generate by their action, like blunt end or sticky end and the susceptibility of the enzyme to the chemical modification of bases like methylation which inhibits the enzyme activity. The exact sized fragments are recovered by using either agarose gel purification technique or sucrose gradient technique which is then ligated to suitable vectors. λ phage, yeast artificial chromosome are considered as suitable vectors for larger DNA and λ replacement vectors like λDASH and EMBL3 are the preferred vectors in constructing genomic DNA library. T4 DNA ligase is used to ligate the selected DNA sequence into the vector.

Construction of cDNA library: Developing cDNA library involves 4 steps. 1. Initial extraction and purification of mRNA, 2. Production of cDNA, 3.Treating the ends of cDNA and 4. Ligation of the cDNA to the vector. The polyadenylated nature of the eukaryotic mRNA enables easy isolation of mRNA by using oligo(dT). Magnetic beads with oligo(dT) is added to the cell lysate which enables the binding of the poly A tail of the mRNA to the oligo(dT) and using strong magnetic force the binded mRNA is isolated from the total RNA. The recovered mRNA is checked for its integrity by using gel electrophoresis technique. Also translation of isolated mRNA is carried out as a step to check its integrity.

Following mRNA extraction begins the production of cDNA. Single strand is produced using mRNA as template by the action of reverse transcriptase enzyme along with addition of 4 dNTPs with oligo(dT) as primer. The enzyme Terminal transferase functions by adding nucleotides to the 3’ end, following which is the elimination of the mRNA strand by alkali treatment. Then the synthesis of second strand begins by the action of reverse transcriptase or klenow polymerase using oligo(dG) primer. This results in the formation of duplex cDNA with extended 3’ end of the strand 1 beyond the 5’ end of the strand 2. Single strand specific nucleases are used to treat the extended 3’ end and as a result any missing nucleotide at the 3’ end is filled by using Klenow polymerase I and dNTPs. cDNA is methylated to ensure the protection from the action of restriction enzymes before adding linkers to the 5’ phosphorylated ends with the help of T4 DNA ligase and ends are made sticky with help of EcoRI enabling the cDNA to ligate with the suitable vector. The frequently used vectors are the plasmids and T4 DNA ligase is used to ligate the cDNA with the vector.

[brianrey]

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[ajithnandanam]

Creating cDNA Library involves the following Steps:

Isolation / Extraction of mRNA

Construction of cDNA / complementary DNA

For the synthesis of cDNA from mRNA poly A tail is used as priming site, a short tag of oligo dT with a free 3'OH group will bind and which will be extended by reverse transcriptase to create cDNA. Next job is to remove the mRNA which is achieved by treating it with RNase enzyme resulting in the single stranded cDNA. The single stranded cDNA need to be converted to double stranded cDNA which is achieved with the help of DNA polymerase. The free 3'OH group for polymerase extension is provided by the single stranded cDNA itself by forming a hairpin loop like structure, which can later be cleaved using nuclease.

Restriction endonucleases and DNA ligase are then used to clone the sequences into bacterial plasmids. The cloned bacteria are then selected, commonly through the use of antibiotic selection. Once selected, stocks of the bacteria are created which can later be grown and sequenced to compile the cDNA library.

[SagarikaGhosh]

Properties of cDNAs and cDNA libraries:

- Eukaryotic cDNAs are free from intron sequences
- They are smaller in size than the corresponding genes, i.e., the genes that encoded them
- A comparison of the cDNA sequence with the corresponding genome sequence permits the delineation of intron/ exon boundaries
- The contents of cDNA libraries from a single organism will vary widely depending on the developmental stage and the cell type used for preparation of the library. In contrast, the genomic libraries will remain essentially the same irrespective of the developmental stage and the cell type used.
- A cDNA library will be enriched for abundant mRNAs, but may contain only a few or no clones representing rare mRNAs.

Subtracted cDNA Library:

When a cDNA library is generated by first removing those mRNA or cDNA sequences that are common to two sources, e.g., two different cell types, such a library is called subtracted cDNA library. Such a library is enriched for those mRNA sequences that are expressed in target cell type, but not for those that are expressed in the other tissue, which is used as the source mRNA or cDNA that is used for subtraction.

Applications of cDNA Library:

Use of cDNA is absolutely essential when the expression of an eukaryotic gene is required in a prokaryote, e.g., a bacterium. The gene expression may be needed either
1. for detection of the clone or
2. the polypeptide product may be the primary objective of cloning.

This is because eukaryotic genes have introns, which must be removed from their transcripts to yield mature mRNAs. Bacteria do not possess the enzymes necessary for removal of introns; therefore, they do not have the ability to produce mature ready-to-translate mRNA molecules from the transcripts of complete native eukaryotic genes. In contrast, functional mRNA molecules do not have introns; hence their cDNA is also free from introns and can be cloned and expressed in bacteria. For example, cDNA for interferon, blood clotting factor VII C (both human) and several other mRNAs have been expressed in bacteria. In addition, cDNAs are used to generate expressed sequence tags (ESTs), which are very useful in high throughput genome research.