CONSTRUCTION OF c DNA LIBRARY KAMAL KUMAR GUPTA
CONSTRUCTION OF c. DNA LIBRARY KAMAL KUMAR GUPTA
c. DNA LIBRARY • A library consisting only of those DNA sequences that are transcribed into m. RNA. • This is because all DNA in this library is complementary to the m. RNA.
PROCEDURE
• A special column contains short oligo (d. T) chains linked to cellulose. • m. RNAs have poly (A) tails. Total cellular RNA is isolated from cell and passed through the column. • The poly (A) tails of m. RNA molecules pairs with the oligo (d. T) chains and the m. RNA is retained in the column whereas the rest of the RNA passes through. • The m. RNA can then be washed from the column by adding a buffer that breaks the hydrogen bonds between the poly(A) tails and the oligo(T) chains leaving only m. RNA with poly(A) tails.
HOW m. RNA CONVERT DOUBLE STRANDED c. DNA ?
• m. RNA molecule copied into c. DNA by reverse trancriptase. • Short Oligo (d. T) primers are added which anneal to the poly(A) tails of the m. RNA and provide 3’OH groups for DNA synthesis • Reverse trancriptase synthesizes a DNA strand by using the m. RNA as a template. • The RNA-DNA hybrid molecule is briefly treated with RNase , which partly digests the RNA strand.
• DNA polymerase is used to synthesise the second DNA strand by using short undigested RNA pieces as primers and the nicks in the sugar-phosphate backbone are sealed by DNA ligase.
ADVANTAGES • It is enriched with fragments from actively transcribed gene. • Intron do not interrupt the cloned sequences. • c. DNA libraries are very useful for eukaryotic gene annalysis.
DISADVANTAGES • It contains only those sequences that are present in mature m. RNA. • Introns and any other sequences that are altered after transcription are not present ; sequences such as promotors and enhancers that are not transcribed into RNA are not present in c. DNA library.
• No c. DNA library was made from prokaryotic m. RNA. • Prokaryotic m. RNA is very unstable • Genomic libraries of prokaryotes are easier to make and contain all the genome sequences.
AN IMPORTANT NOTE • c. DNA library represents only those gene sequences expressed in the tissue from which the RNA was isolated. • Frequency of a particular DNA sequences in the c. DNA library depends on the abundance of corresponding m. RNA in the given tissue.
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