C dna is made from a mature m rna of eukaryotic cell with the utilization of enzyme called as reverse transcriptase.
Steps in c dna creation
1. m rna is made and purified from remaining of the rna.
2. The m rna purified is tagged by oligo d t nucleotide as a complementary primer which binds with pol a tail of m rna developing a free 3"oh end that can be extended through reverse transcriptase to develop a complementary dna strand. Now the m rna is eliminated through rnase enzyme leaving a single stranded c dna. This single stranded c dna is changed to double stranded dna with help of dna polymerase.
Diagram in the attachment
The sanger technique, also known as dideoxynucleotide sequencing or chain termination sequencing, is based on the utilization of dideoxynucleotide (ddntp) in addition to normal nucleotides (dntp) found in c dna. Dideoxynucleotide are basically the same as nucleotides apart from, they include a hydrogen group on the 3' carbon inspite of a hydroxyl group (oh). These modified nucleotides, when incorporated into a cdna sequence, stop the addition of additional nucleotides therefore ends the elongation of the dna chain. This takes place because of phosphodiester bond cannot made between the dideoxynucleotide and the other incoming nucleotide, and therefore the dna chain is terminated.
Steps in sanger technique of cdna sequencing
1. The area of cdna to be sequenced is amplified and then denatured to create single stranded dna.
2. A sequencing primer is annealed to form single stranded dna.
3. Dideoxynucleotide chain termination dna sequencing then gets advantage of the fact that a increasing chain of nucleotides, extending in 5' to 3' direction, will end if, instead of a conventional deoxynucleotide, a 2'3' dideoxynucleotide gets incorporated. By doing four different reactions, each involving a dna polymerase and small amount of one of the four dideoxynucleotides in addition to four deoxynucleotides, four different sets of chain-terminated fragments can be created.
4. Subsequent to the replication/termination step, these chain terminated fragments will stay bound to the single stranded dna molecule which has acted like a template. Then heating these partially double stranded molecules and adding a denaturing agent like formamide, the single stranded chain termination molecules can be free from their template and separated utilizing high resolution denaturing gel electrophoresis.
5. The sequence of original region of cdna is then ultimately deduced through examining the associated positions of the dideoxynucleotide chain termination products in the four lanes of the denaturing gel.