BLOSUM matrices were first introduced in a paper by Steven Henikoff and Jorja Henikoff.[1] They scanned the BLOCKS database for very conserved regions of protein families (that do not have gaps in the sequence alignment) and then counted the relative frequencies of amino acids and their substitution probabilities. Then, they calculated a log-odds score for each of the 210 possible substitution pairs of the 20 standard amino acids. All BLOSUM matrices are based on observed alignments; they are not extrapolated from comparisons of closely related proteins like the PAM Matrices.