Just one place and one point separated the two teams before the Championship contest at the Madejski stadium.
But it was Reading who appeared the superior side in a dominant opening and they got their reward when Mackie seized on a loose ball before drilling home from 18 yards in the 23rd minute.
Brighton found a way back into the game in the 53rd minute when Chris O'Grady levelled from the spot after Jake Cooper was adjudged to have felled Emmanuel Ledesma in the penalty area.
However, three minutes later Reading were back in front when Mackie let fly with an unstoppable left-foot effort from 25 yards and that proved enough for Steve Clarke's men to move up to 16th.
In League One, leaders Bristol City's march towards promotion showed no signs of stopping as they comfortably won 3-0 at rock-bottom Yeovil Town.
All of the goals came in the second half, as Kieran Agard, Luke Freeman and George Saville all got their names on the scoresheet at Huish Park.
Rounding off the action, relegation-threatened Coventry City let the lead slip in a 1-1 draw at home to Bradford City.
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