Dufner shot a second successive seven-under-par round of 65 for a tournament record 36-hole total of 14 under par.
Rickie Fowler shot six under for the day to move third and within one stroke of fellow American Daniel Summerhays, with Jordan Spieth still on six under.
Scotland's Martin Laird is a stroke further back at five under par.
Dufner, who began his second round on the 10th, holed from 176 yards at 18 as his approach to the back of the green rolled down the slope and into the cup for an eagle.
"We got two more rounds to go. We'll see how the course changes, if the wind picks up, gets running fast, it plays different. We'll just see," said the 40-year-old.
World number six Spieth was one shot off the lead after an opening round of 66, but three bogeys on the front nine saw him drop to tied fourth.
Meanwhile, world number one Dustin Johnson missed the cut at eight over par.

John Lamont quit the Ettrick, Roxburgh and Berwickshire constituency seat to fight for a place at Westminster, hoping to unseat the SNP's Calum Kerr.
The Conservative MSP formally resigned from the seat as of 13:00 on Thursday.
The by-election means residents in the area will vote on their local council, their Holyrood representative and their MP all inside five weeks.
Selection procedures for the Holyrood by-election have yet to get under way.
Presiding Officer Ken Macintosh has written to Tracey Logan, chief executive of Scottish Borders Council and returning officer for the elections, to confirm his choice of date and to ask for arrangements for the by-election to be put in place.
Mr Lamont is seeking to unseat the SNP's Calum Kerr in Berwickshire, Roxburgh and Selkirk, Scotland's most marginal Westminster constituency. Mr Kerr gained the seat from Lib Dem former Scottish Secretary Michael Moore in 2015 by a margin of 328 votes.
The Lib Dems have put forward Catriona Bhatia, currently deputy leader of the council, as their candidate for the Westminster poll.

Brenda Wardle - who denies the charges - is accused of pretending to be a practising attorney and offering to represent a convicted murderer.
She appeared at Port Elizabeth Commercial Crimes Court on Monday, South African media report.
She featured as an expert on the BBC during the 2014 Pistorius trial.
Ms Wardle, 56, also boasts of appearing on Sky News, Fox News and a number of South African broadcasters on her social media profiles.
She has published a book called To Kill A Fragile Rose: The State's Case Against Oscar Leonard Carl Pistorius.
According to local media, these allegations date back to 2009, when she offered to represent a convicted murderer.
The family accuse her of charging around 500,000 rand (£28,600; $36,800) for her services over the next four years.
Ms Wardle will represent herself, against the advice of the magistrate, The Herald reports.
She was refused bail after failing to arrive at court on several occasions, IOL News said.
Ms Wardle will next appear in court on 27 July.
American Jason Dufner will take a five-shot lead into the third round of the Memorial Tournament after carding an eagle on the 18th hole on Friday.
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A Holyrood by-election is to be held on the same day as the UK's snap general election on 8 June.
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A woman who acted as a legal analyst for the media during the Oscar Pistorius trial has appeared in court charged with fraud.