Cloning in lambda vectors

preparation of vector

– digested with restriction endonuclease

– substitution vectors often purified by size

– separates stuffer fragment

– unless lambda EMBL type vector

– use sucrose gradient or agarose gel

– sucrose gradient often preferred

– no polysaccharides interfere with digests

alkaline phosphatase

– enzyme that removes 5' PO₄ from DNA

– prevents self ligation

(1 molecule in ligation must have 5' PO₄)

2 uses – treat vector – prevents religation of lambda vector arms

– important for insertion vector

– vector can be packaged without insert

– treat cloned DNA

– stops concatemers of short DNA fragments

– inserted in vector

– could produce false genomic maps

Note: – cannot do both

– one molecule must have 5' PO₄ for ligation

preparation of cloned DNA

– genomic library

– usually want overlapping 15-20 kb fragments

– right size to fit in substitution vector

– overlapping fragments allow mapping

– “chromosome walking”

choice of restriction endonucleases

– 6-cutter – e.g. BamHI

G\GATCC

CCTAG\G

– will cut on average every 4⁶ bp - ~ 4 kb

– but cannot count on this

– e.g. with repeated DNA sequences

– may get no cutting for > 20 kb

– too long for many vectors

– fragment not packaged

– 4-cutter – e.g. Sau3A

\GATC

CTAG\

– will cut on average every 4⁴ bp – 256 bp

– essentially random cutting throughout genome

– can do partial digest

– use insufficient enzyme and/or time for complete digest

– get mix of overlapping DNA molecules of varying sizes

– will contain most of genome

– need about 3-5 genomes worth of clones to be sure

– statistical odds of getting every fragment

– also increases chances of overlapping clones

– e.g. for human genome – 3 X 10⁶ kb

– to clone entire genome in 15 kb fragments

– need 2 X 10⁵ clones X 3-5 = 6 X 10⁵- 1 X 10⁶ clones

Sau3A

– has matching 5' “sticky ends” with BamHI cut vector

– fragments can be cloned in lambda EMBL type vector

– and subcloned with SalI sites

– Sau3A/BamHI hybrid sites

– only BamHI sites ¼ of time

– Sau3A cuts too often for subcloning

size selection – lambda vectors

– allow selection of size fragments cloned

– minimum/maximum distance between cos sites

problem: – concatemers of short fragments

– may produce packageable product

– incorrect map

solutions: – can use phosphatase on cloned DNA

– no ligation between cloned fragments

– can isolate DNA fragments of desired size

– use sucrose gradient or agarose gel

– sucrose gradient often preferred

– no polysaccharides interfere with digests