cDNA library

Source: Wikipedia, the free encyclopedia.

A cDNA library is a combination of cloned cDNA (

introns, and other regulatory elements found in a genomic DNA library.[1]

cDNA Library Construction

Formation of a cDNA library.

cDNA is created from a mature

poly-A tail.[2]

mRNA extraction

Firstly, mRNA template needs to be isolated for the creation of cDNA libraries. Since mRNA only contains exons, the integrity of the isolated mRNA should be considered so that the protein encoded can still be produced. Isolated mRNA should range from 500 bp to 8 kb.

column purification. Column purification can be done using oligomeric dT nucleotide coated resins, and features of mRNA such as having a poly-A tail can be exploited where only mRNA sequences containing said feature will bind. The desired mRNA bound to the column is then eluted
.

cDNA construction

Once mRNA is purified, an oligo-dT primer (a short sequence of deoxy-thymidine nucleotides) is bound to the poly-A tail of the RNA. The primer is required to initiate DNA synthesis by the enzyme

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.

cDNA Library uses

cDNA libraries are commonly used when reproducing eukaryotic genomes, as the amount of information is reduced to remove the large numbers of non-coding regions from the library. cDNA libraries are used to express eukaryotic genes in prokaryotes. Prokaryotes do not have introns in their DNA and therefore do not possess any enzymes that can cut it out during transcription process. cDNA does not have introns and therefore can be expressed in prokaryotic cells. cDNA libraries are most useful in reverse genetics where the additional genomic information is of less use. Additionally, cDNA libraries are frequently used in functional cloning to identify genes based on the encoded protein's function. When studying eukaryotic DNA, expression libraries are constructed using complementary DNA (cDNA) to help ensure the insert is truly a gene.[4]

cDNA Library vs. Genomic DNA Library

cDNA library lacks the non-coding and regulatory elements found in genomic DNA.

Genomic DNA libraries
provide more detailed information about the organism, but are more resource-intensive to generate and keep.

Cloning of cDNA

cDNA molecules can be cloned by using restriction site linkers. Linkers are short, double stranded pieces of DNA (oligodeoxyribonucleotide) about 8 to 12 nucleotide pairs long that include a

sticky end" ligation of the insert into the vector the resulting recombinant DNA molecule is transferred into E. coli
host cell for cloning.

See also

References

  1. ^ "cDNA Libraries - an overview | ScienceDirect Topics". www.sciencedirect.com. Retrieved 2024-02-19.
  2. ^ cDNA library || How cDNA library is constructed? || What are DNA libraries used for?, retrieved 2024-02-19
  3. ^
    S2CID 25600775
    .
  4. ^
    OCLC 226038060.{{cite book}}: CS1 maint: multiple names: authors list (link
    )

External links