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by the end of this a level biology revision lesson you will: -
1. Know that the enzyme reverse transcriptase polymerises DNA from an RNA template.
2. Explain the steps involved in producing a cDNA clone from a mRNA strand of interest.

In the Previous Lesson we outlined the key principle of what recombinant DNA is, the knowledge required to understand DNA technology (i.e. understanding transcription, translation, the use of particular enzymes etc.).

In this lesson we will focus on how DNA clones, called complementary DNA (cDNA) can be produced by making use of the enzyme Reverse Transcriptase. You’ll Remember now, that Viruses, called Retroviruses (like HIV) have enzymes called Reverse transcriptase. These Enzymes are able to “reverse transcribe” RNA into DNA.

This technique utilises reverse transcriptase enzymes to make a complementary DNA (cDNA) fragment - specifically of the Gene of interest from a mRNA molecule.

You know that mRNA is required to direct the synthesis of a polypeptide chain, and that there are lots of mRNAs produced during transcription… in order to make lots of the required protein… So it stands to reason that it must be easier to acquire a mRNA molecule from the cell, for the protein we want to make – than it is to acquire the specific DNA section (the gene of interest) directly from the chromosome itself… Since of course there are only 2 copies of the gene in the first place (one at each locus, on each homologous chromosome).

Now, since there are many mRNAs being produced from the transcription of the gene we’re interested in… we just need a way to “reverse” the mRNA code back into the DNA code from which it was transcribed… and that is where we make use of Reverse Transcription Enzymes (acquired from retroviruses).

Reverse transcriptase directs the synthesis of a DNA strand from an RNA template.

Here are the steps you need to know about producing a DNA section (gene of interest) from mRNA obtained from cells producing a protein we want to make…

1. Extract mRNA from a cell (e.g. if we want to make the protein Insulin, we can isolate and extract mRNA (transcribed from the gene that codes for the production of insulin) from a pancreatic cell.
2. Add “Poly T” primers to hybridise (complementary base pair) to the poly A tail of mRNA.
3. Reverse transcriptase copies the mRNA forming a cDNA / mRNA “hybrid”
4. The cDNA / mRNA hybrid is degraded (its hydrolysed, split apart – using an enzyme called RNase H) [you don’t need to recall the name of this enzyme]!
5. DNA polymerase is added, which synthesises double stranded cDNA (from the cDNA template).

Now, we are able to take the double stranded cDNA and insert it into a virus vector or a plasmid, so we can make lots of copies of that cDNA molecule – the result is a collection of “cloned cDNA” molecules… the cDNA clones obtained from a single mRNA are collectively known as a cDNA library…

And that is how we use Reverse transcriptase to create a copy of DNA – for the gene we are interested in from mRNA molecule…

A Level biology Exam style question.

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