Base pairing between adenine and thymine can be found in DNA only. There are two hydrogen bonds holding the two nitrogenous bases together. One of the hydrogen bonds is formed between one of the Hydrogen atoms of the amino group at C-6 of adenine and the Oxygen atom of the keto group at C-4 of thymine. Another bond is found between Nitrogen atom at position 1 of adenine and Hydrogen atom linked to N-3. The hydrogen bonds between adenine and thymine are important for DNA to maintain a double helix structure. Since they are not very strong bonds, they can be broken at elevated temperature. In DNA replication and transcription, the initiation of these reactions often starts at A-T rich sites because the breakage of two hydrogen bonds between A and T requires less energy than G-C rich sites which have three hydrogen bonds between G and C.

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Cytosine and guanine pairing can be found in both DNA and DNA-RNA hybrid formed during replication and transcription. The two nitrogenous bases are held together by three hydrogen bonds. The first hydrogen bond is found between Oxygen atom of the keto group at C-2 of cytosine and one of the Hydrogen atom of the amino group at C-2 of guanine. The second hydrogen bond is formed between N-3 of cytosine and Hydrogen atom attached to N-1 of guanine. The interaction between Hydrogen atom of the amino group at C-4 of cytosine and Oxygen atom of keto group at C-6 of guanine is the third hydrogen bond.  DNA with higher G-C content is more stable than DNA with  A-T rich regions. Having one more hydrogen bond between G-C than A-T needs more energy to break the nitrogenous bases apart. Thus, the melting temperature is relatively higher when DNA has higher C-G content. C-G content is used to predict the annealing temperature of primer to DNA in polymerase chain reaction.

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