All are steps of PCR EXCEPT:

ANSWER: (D) TransformationREF: Harper's 28 ed chapter 39The polymerase chain reaction (PCR) is a method of amplifying a target sequence of DNA. PCR provides a sensitive, selective, and extremely rapid means of amplifying any desired sequence of DNA. Specificity is based on the use of two oligonucleotide primers that hybridize to complementary sequences on opposite strands of DNA and flank the target sequence. The DNA sample is first heated to separate the two strands of the template DNA containing the target sequence; the primers, added in vast excess, are allowed to anneal to the DNA; and each strand is copied by a DNA polymerase, starting at the primer sites in the presence of all 4 dXTPs. The two DNA strands each serve as a template for the synthesis of new DNA from the two primers. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences, and extension of the annealed primers withDNA polymerase result in the exponential amplification of DNA segments of defined length (a doubling at each cycle). Early PCR reactions used an E coli DNA polymerase that was destroyed by each heat denaturation cycle. Substitution of a heat-stable DNA polymerase from Thermusaquaticus (or the corresponding DNA polymerase from other thermophilic bacteria), an organism that lives and replicates at 70-80degC, obviates this problem and has made possible automation of the reaction, since the polymerase reactions can be run at 70degC. This has also improved the specificity and the yield of DNA.