6. COMPLEMENTARY DNA (cDNA)
Complementary DNA (cDNA) is derived from RNA - usually messenger RNA (mRNA).
Most genes are single copy in DNA (exception: ribosomal 5S gene in oocytes).
mRNA is often more abundant (i.e. present in multiple copies) in certain cells than the, usually, single-copy DNA gene This is assuming that the gene is being expressed in the cells at the time. Therefore, it is often easier to find the gene in its mRNA form.
e.g. insulin mRNA in b cells of Islets of Langerhans in the pancreas; albumen mRNA in oviduct cells; antibody mRNA in plasma cells.
However, if the gene is from a eukaryote, it will contain introns which are spliced out before mRNA is translated and so the gene may be in a different form from the original DNA form.
mRNA cannot be manipulated directly (restriction endonucleases only act on double-stranded DNA) and, therefore, mRNA has to be converted into DNA. This is done using RNA as the template and the enzyme reverse transcriptase which is obtained from retroviruses. This is the reverse of 'normal' transcription where the template is DNA and the product mRNA.
There are several methods of synthesizing cDNA. The method that follows is one example and exploits the fact that eukaryotic mRNA has a tail of adenine bases at the 3' end which anneals with an oligo-dT primer (required to initiate strand synthesis). The second DNA strand is synthesized using another primer (oligo-dG) which anneals to cytosines previously added to the 3' end by a terminal transferase enzyme.
SYNTHESIS OF COMPLEMENTARY DNA (cDNA)
USES OF cDNA:
1. For nucleotide (base) sequencing and gene analysis.
2. For production of probes to target chromosomal genes.
3. For insertion into host-vector systems for cloning and/or expression (perhaps of useful products).
4. For construction of cDNA libraries (collections of random fragments of cDNA which contain all the mRNA being transcribed by the source cells at the time).
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OF SECTION 6.
NOW GO TO
SECTION 7
(GENE PROBES).
A sequence of a gene which is transcribed but which is excised by a splicing reaction before the mature mRNA is translated. Common in eukaryotes, but rarer in prokaryotes.