BLAST-OER

Comparing Sequences

There are several applications in biomedical science that need to compare DNA, RNA or protein sequences:

trying find a matching sequence in a database for a sequence you found in an experiment or
comparing two sequences to determine the similarity and possible evolutionary relationship. This comparison is done by the basic local alignment search tool (BLAST).

The first step of BLAST in any of these comparisons is to find the best alignment.

This sequence alignment is done by (pairwise) sequence alignment, defined as the process of arranging two sequences to achieve maximal levels of identity and conservation.

Identity: A residue is exactly the same (either nucleotide or amino acid)
Conservation: An amino acid is replaced by another amino acid that preserves the physicochemical properties of the original residue (for nucleotides, conservation has no meaning, as due to the triplet code translation, two DNA/RNA residues can only be identical or not for the meaning of the code)
Similarity: The resulting extent to which sequences are related, based on identity and conservation.

For conservation, the group to which an amino acid belongs is important (basic, acidic, uncharged polar, or nonpolar side chain), as well as the size of the amino acid’s side chain. Also, some amino acids have very unique properties, making them essential in certain positions in a protein (e.g. cysteine, to form sulphur bridges).

Homology is defined as similarity attributed to descent from a common ancestor. Sequences are either homologous or not (they cannot be somewhat homologous due to similar protein domain function). Orthologues are homologues between species (e.g. human and mouse haemoglobin alpha chain), paralogues are homologues within a species (e.g. human haemoglobin alpha and beta chain).

Using sequence alignment allows us to assess the degree of similarity and the possibility of homology.

Besides checking identity and conservation of residues, we also should be able to account for the occurrence of (small) insertions and deletions. This is done by the concept of a gap: when aligning two sequences, each of them can be broken apart, and some residues in the other sequence can be matched to empty spaces in that sequence.

Comparing protein or nucleotide sequences?

Alignment of protein sequences can be more informative than alignment of nucleotide sequences. This has several reasons:

There are 20 different amino acids, but only 4 different nucleotide bases
For amino acids we can take their biophysical similarities into account
Codons are degenerate: changes in the third position of coding sequences often do not alter the amino acid that is specified

So, protein sequences offer a longer “look-back” time.

This means that it can be more useful to use protein sequences than DNA or RNA sequences, especially when the expected divergence between the sequences is high (e.g. large evolutionary distance).

Alignment of nucleotide sequences is still useful, for example to:

study noncoding regions of DNA (promoter regions, intergenic regions)
confirm the identity of a cDNA
study DNA polymorphisms
study genomes (compare genomes, create genome annotations)

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