I'm assuming that the original poster wanted the frequency of all pairs, so we don't want the regex engine to consume more than one character per iteration. With...

$genome="acgt";
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Then the look ahead version will finish with...

$count{ac} == 1
$count{cg} == 1  #Note this match
$count{gt} == 1
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...while the non-lookahead version will have...

$count{ac} == 1
$count{gt} == 1
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... i.e. it misses the "cg" case because it jumps through the string two characters at a time.