a Copy DNA Library from mRNA
you know what the product of your gene is, say for example
you want the piece of DNA that codes for insulin; you would
expect to find lots of mRNA for this protein in the cells
of the pancreas. These cells specialise in making insulin
and thus contain large amounts of mRNA transcribed from the
make a cDNA library: -
First extract all the mRNA from the specialised cells by cell
fractionation and purification.
mRNA sample can now be used to make cDNA (copy DNA) in a process
that is the reverse of transcription.
Transcription is the process where mRNA is made as the enzyme
RNA polymerase feeds along the DNA sequence coding for the
gene in the nucleus of the cell.
Reverse transcription doesn't occur in normal eukaryotic cells
and requires the enzyme Reverse Transcriptase.
enzyme is only made by retroviruses like the HIV virus, which
can carry out transcription backwards.
the mRNA with the Reverse Transcriptase and a mixture of DNA
nucleotides and a complementary DNA strand is built up on
the mRNA template.
will still be many different types of mRNA template obtained
from a specialised cell and the one you want will need to
be identified using a probe, in exactly the same way as for
the Piece you want.
this process has one big advantage over a Genomic DNA
library. A cDNA library will contain a bigger proportion of
the required gene, because the mRNA was taken from a cell
containing a lot of the required protein.