cDNA

What’s a cDNA :

It's the DNA copy of a mRNA without intron obtained by reverse transcriptase. cDNAs have no introns and can be expressed by the bacteria.

Synthesis of cDNA :

Manipulation and study of RNA is difficult because of their high sensitivity to ribonucleases that destroy, so :

- It is necessary to recopy the sequence to DNA to be more stable and that can be amplified as required.

- The purified mRNA (affinity chromatography on an oligo-d(T) column) is bonded at first with oligonucleotides poly (T) which will be attached to the poly (A) tail.

- From the 3 'end of this primer poly (T) the reverse transcriptase, which is a DNA polymerase, synthesizes a DNA strand complementary to the messenger of departure.

- Once completed this synthesis the RNA is degraded by a specific ribonuclease or by a strong base.

- The DNA strand made ​​spontaneously forms at its 3'end of a hairpin loop by hybridizing to itself.

- The 3 'end of the loop will serve as a site of start for the DNA polymerase which will synthesize a complementary DNA strand of the first.

- A specific nuclease of single-stranded DNA removes the loop from the end. The double-stranded cDNA is ready.

- Thereby we can constitute much of cDNA that it exists messengers in a cell: all of these cDNA form a «cDNA library».

 

Synthesis of cDNA