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Making a Copy DNA Library from mRNA

If 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 Insulin gene.

To make a cDNA library: -

1. First extract all the mRNA from the specialised cells by cell fractionation and purification.

This mRNA sample can now be used to make cDNA (copy DNA) in a process that is the reverse of transcription.

REMEMBER;- 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.

2. Reverse transcription doesn't occur in normal eukaryotic cells and requires the enzyme Reverse Transcriptase.



This enzyme is only made by retroviruses like the HIV virus, which can carry out transcription backwards.

 

Incubate the mRNA with the Reverse Transcriptase and a mixture of DNA nucleotides and a complementary DNA strand is built up on the mRNA template.

3.There 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 a Genomic DNA library. See Finding the Piece you want.

However, 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.