Sequencing longer distances

– traditional sequencing gels – 200 bp per loading

– bands near top not well separated

– cannot be read accurately

solutions:

double loading – divide reactions in ½ after extension

– load ½ of reactions & run into gel

– load rest in second loading ~2 hours later

– first loading run off bottom of gel

– short fragments lost

– but longer fragments separated

– short fragments still visible in second loading

wedge gels – wedge-shaped side spacers

– cause difference in voltage over length of gel

– more even spread of bands

electrolyte gradient gels

– same effect by varying concentration of buffer across gel

– dye terminator sequencing

– bands read continuously through run

– above techniques not needed

– 400 bases (standard) to >600 bases (best equipment)

How can fragments longer than 0.5-1 kb be sequenced?

– can sequence from ends using universal primers

– make new primers from end of known sequence

– use to sequence farther

– expensive (multiple primers) & time-consuming

– can make sets of deletions within cloned fragment

– use exonuclease III

– digests 1 strand of DNA from 3' end

– only if end blunt or 5' extension

– 3' extension not digested

– cut at one end of clone

– using 2 REs in multiple cloning site

– 1 is blunt or 5' (clone side)

– 1 is 3' extension (vector side)

– digest with exonuclease III for different times

– get different amounts of deletion

– digest remaining single strand using S1 nuclease

– digests single-stranded DNA only

– converts both ends to blunt – T4 DNA polymerase

– ligate – get set of clones with various-sized deletions

– vector not deleted – sequence all clones & align overlaps