Why does double-stranded DNA with a high A-T content have a lower transition temperature, Tm, than double-stranded DNA with a high C-G content? The fewer H-bonds for A-T-rich dsDNA cause its two strands to stick together less well than G-C-rich dsDNA. A-T-rich dsDNA requires more energy to separate into single strands than G-C rich dsDNA. Elevated temperature breaks the backbone bonds between C and G bases less easily than they break the backbone bonds between A and T bases. Purine bases are more susceptible to hydrolysis at high temperature than are pyrimidine bases.

DNA is a double helix in which two DNA strands are held together with the help of hydrogen bonds formed between their complementary bases. Adenine of one DNA strand always forms two hydrogen bonds with thymine of other DNA strands. Likewise, guanine of one DNA strand pairs with cytosine of other DNA strands by the formation of three hydrogen bonds.

The presence of lesser hydrogen bonds in AT base pairs makes an AT-rich DNA double helix to melt at lower temperatures. On the other hand, a GC rich DNA double helix has a higher melting point as more amount of energy is required to break three hydrogen bonds per base pair as compared to only two H-bonds per AT pair.