Nucleic Acid Hybridization
Hybridization is the process of complementary base pairs binding to form a double helix. Melting is the process by which the interactions between the strands of the double helix are broken, separating the two nucleic acid strands. These bonds are weak, easily separated by gentle heating, enzymes, or physical force. Melting occurs preferentially at certain points in the nucleic acid. T and A rich sequences are more easily melted than C and G rich regions. Particular base steps are also susceptible to DNA melting, particularly T A and T G base steps. These mechanical features are reflected by the use of sequences such as TATA at the start of many genes to assist RNA polymerase in melting the DNA for transcription.
Strand separation by gentle heating, as used in PCR, is simple providing the molecules have fewer than about 10,000 base pairs (10 kilobase pairs, or 10 kbp). The intertwining of the DNA strands makes long segments difficult to separate. The cell avoids this problem by allowing its DNA-melting enzymes (helicases) to work concurrently with topoisomerases, which can chemically cleave the phosphate backbone of one of the strands so that it can swivel around the other. Helicases unwind the strands to facilitate the advance of sequence-reading enzymes such as DNA polymerase.
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