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».
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| Synthesis of cDNA |
