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

DNA polymerases

DNA polymerases (or deoxynucleotidyl transferase) are the enzymes responsible for the polymerization of nucleotides during DNA replication. They are dependent on DNA, that is to say, they need a DNA template to produce the newly synthesized strand, and for this they read the template strand of 3 'to 5' to synthesize DNA in the 5’ to 3'.

Prokaryotic DNA polymerases are 3 types (I, II and III) and eukaryotic DNA polymerases are 5 types (α, β, δ, ε and γ).

DNA polymerases require a number of conditions of activities:

- The four deoxyribonucleotides 5' triphosphate (dATP, dTTP, dCTP and dGTP) in equimolar amounts.

- Magnesiums ions (Mg2 +) which stabilize DNA and proteins.

- A template DNA (single or double stranded).

- A DNA or RNA primer with a free 3' OH end.

Activities of DNA polymerases:

During the formation of a phosphodiester bond between deoxyribonucleotides 5' triphosphate and the strand being elongation, there is hydrolyzed to the function triphosphate and formation of pyrophosphate (PPi).

DNA polymerases have very specific activities:

- A polymerase activity 5' to 3' which is their main activity.

- Exo-nuclease activity correspondes in the degradation of one end of the newly synthesized strand of the DNA during replication and which can be of 2 types:

        -From 3' to 5', which corresponds to the degradation from the 3' OH end. The exonuclease activity 3' to 5' allows so-called proofreading, which corresponds to the correction of a mismatch base by breaking the phosphodiester bond and replacing the mismatched nucleotide.

        - From 5' to 3', which corresponds to the degradation from the 5 'phosphate end, at the junction of the DNA segments of the lagging strand synthesized.