Steps of C dna amplification
1. Smart-pcr amplification of cdna is the technique which initiates with the change of mrna to cdna utilizing mmlv-rt, mutated in the rnase h domain, in the first strand synthesis reaction. This enzyme owns reverse transcriptase activity, dna-dependent dna polymerase activity and terminal deoxynucleotidyl transferase (tdt) activity. In first strand synthesis, an anchored oligo-d(t) primer (3' cdna synthesis primer) anneals to the 5'end of poly(a) tails. The 5'-end of this 3' cdna synthesis primer includes a defined sequence that gives as a pcr primer target site in the corresponding amplification step. When the first strand has been elongated to the end of an mrna template, the tdt action of the reverse transcriptase adds various un-templated deoxycytosine (d(c)) residues at the 3'-end of first strand cdnas (in a fashion similar to the addition of single un-templated d(a) residues to the end of pcr products produced with taq dna polymerase). The 5'cdna synthesis primer includes three guanosine residues at its 3' end that anneal to these un-templated d(c) "tails"
And then provides as a template for the dna-dependent dna polymerase activity of the mmlv-rt. This "switching mechanism at rna termini" (smart) permit for mmlv-rt-mediated 3'-extension of all first strand cdna products to inolve a defined, contiguous sequence imitated from the 5'cdna synthesis primer. The terminal transferase and template switching activities takes place during the first strand cdna synthesis. Areas of sequence in the 5'cdna synthesis primer and the 3'cdna synthesis primer are similar, permitting for uniform amplification of all first strand cdna with single pcr primer.
2. ssh-pcr depends on principles of dna hybridization. Cdnas made from the two mrna populations being compared are firstly digested with a four base cutting restriction enzyme to create short blunt-ended fragments which are more encouraging to the suppressive pcr activity. One of the cdna populations which is assumed to include an overrepresentation of few unknown genes, is signified as "tester". The tester cdna is divided to two aliquots and each one is ligated to a different adaptor. The other cdna population is signified as "driver" and is mixed with two adaptor-ligated tester cdnas independently. The two mixtures are permitted to undergo denaturation and renaturation. In these complexes, the driver is in surplus and hybridizations do not go to finishing point. Sequences over-represented in the tester cdna pool will remain single stranded and sequences common to both tester and driver reanneal as they nucleate more often because of their higher relative concentrations. In second hybridization step, the single stranded tester sequences left in the first two hybridization mixes, which bear dissimilar adaptor sequences at their ends, are permitted to fully re-anneal in the presence of additional denatured driver cdna. In second hybridization, there is again subtraction of sequences ordinary to both tester and driver cdnas. As these hybridizations go for completion, single stranded fragments exclusive to the tester cdna create hybrid molecules with different adaptor sequences at either end.
The complete population of molecules is then exposed to two rounds of pcr to selectively augment the differentially expressed sequences. Before the first pcr, adapter ends are filled in, therefore creating the complementary primer binding sites required for amplification. Double stranded molecules including only one adaptor-ligated strand (rising from adaptor-ligated sequence annealing to driver cdna is only linearly amplified. Reannealed driver cdnas are not augmented type a and d molecules lack primer annealing sites and are not amplified. Exponential amplification is preferred by molecules with diverse adaptor sequences are either end arising from tester cdna population.
Differentially expressed sequences are highly enriched in type e fraction, and thus in the subtracted cdna pool. This technique does not involve any kind of physical separation of single-stranded molecules from double-stranded hybrids.