Working with large DNA fragments

preparation of DNA: – problem is shearing

– minimize handling of DNA

– e.g. phenol/chloroform, centrifugation, etc.

– lyse DNA in agarose blocks

– mix cells with 1 % agarose, cool in mould

– lyse cells with SDS/proteinase K

– inhibit protease – phenylmethylsulfonyl fluoride (PMSF)

– stops degradation of restriction endonucleases

– digest DNA with restriction endonucleases (RE)

– suspend blocks in digest mix – let digest overnight

– time for RE to diffuse into block

– blocks loaded directly into wells on gel

– DNA electrophoresed into gel

restriction digests – 2 possibilities:

1. partial digests with 6-cutters – e.g. EcoRI, SmaI

– get variety of fragments, different sizes

2. digest with rare-cutting enzymes – e.g. NotI (8-cutter)

GC\GGCCGC

CGCCGG\CG

– cuts ~ every 4⁸ bp – 65.5 kb

– cuts less often in mammalian DNA

– CG sequence underrepresented in mammals

– methylation site – methyl-C

– hotspot for deamination & mutagenesis

pulsed-field gel electrophoresis (PFGE)

– standard electrophoresis

– cannot separate bands larger than 20-40 kb

– larger bands have same motility – smear on gel

– PFGE uses alternating current directions

– tips of DNA molecules to briefly run backward

– bunches DNA up in clump

– must sort out into linear form before moving

– longer molecules take more time to sort out bunch

– retarded movement through gel

– 2 main types:

field-inversion gel electrophoresis (FIGE)

– switch current backwards & forwards in gel

– separates molecules of 10-2000 kb

– can be done in standard gel apparatus

orthagonal field gel electrophoresis (OFAGE)

– uses currents at different angles to gel

– separates Mb-sized pieces

– needs specialized (expensive) equipment

molecular weight markers

lambda ladder – mix of lambda concatemers of various lengths

– multiples of 48 kb

Saccharomyces cerevisiae chromosomes

– from lysed yeast cells (in agar blocks)

– range of bands from 200 kb to 1200 kb

– useful for YAC isolation

– YAC “chromosome”

– is different length from other chromosomes