Restriction Endonucleases (RE)

– bacterial enzymes

– recognise & cut specific DNA sequences

– unless protected by methylation of bases

original purpose – destroy phage DNA

– name comes from “restriction” of phage host range

e.g. lambda grown in E. coli C – infected into wild type E. coli K

– get very low yield of plaques

– reason: E. coli C does not methylate specific site for EcoK enzyme

– phage genomes are cleaved by EcoK enzyme

(produced by E. coli K)

– lambda grown on E. coli K – methylated genomes

– not cleaved when infects other E. coli K cells

3 types of enzyme:

Class I

– methylase & endonuclease part of same enzyme complex

– cut distant (100-1000 bp) from recognition site

Class II – methylase & endonuclease separate enzymes

– (usually) cut within recognition site

(some cut near recognition site)

Class III

– endonuclease requires methylase to recognise site

– cut close (25-27 bp) to recognition site

– Classes I & III – useless for cloning – random ends

– may interfere with cloning foreign DNA

– e.g. E. coli K strains used as plasmid/phage hosts

– have hsd ^– mutation – inactive EcoK complex

Class II – most have specific cut site – cloning tools

may produce 5' or 3' cohesive (sticky) single-stranded ends

– will anneal to matching sticky ends (H-bonding)

ApaI

5' GGGCC/C 3'

3' C/CCGGG 5'

Bsp120I

5' G\GGCCC 3'

3' CCCGG\G 5'

may produce blunt ends

– anneal to other blunt ends (at lower efficiency)

HincII

5' GTPy/PuAC 3'

5' CAPu/PyTG 3'

– sequence may be degenerate (like HincII)

– variable cut sites

isoschizomers – enzymes that cut same site

– may not cut same way

– ApaI 3' ends, Bsp120I 5' ends

frequency of cutting sites

– predicted by number of bases in site

– n bases in site, 4ⁿ bp between sites

problems: repetitive sequences – not random sequence

– GC content of DNA

– if not 50 %, not equal chance for all bases to occur

– “hot spots” for mutagenesis

– underrepresented sequences

restriction endonuclease names

– derived from species, strain, order of discovery

– e.g. EcoRI – first RE found in E. coli strain R

– first 3 letters italicized/underlined, rest not

RE use: REs are expensive and labile

– fresh tip every time enter RE stock

– keep cold

– in freezer, on ice (or freezer block) outside freezer

– if preparing multiple digests

– prepare diluted aliquot first

– dispense to individual reactions

– decreases amount enzyme used

– less chance of contaminating stock

RE activity:

defined in units = 1 µg lambda DNA cleaved per hour

10 µg lambda DNA

– cleaved by 10 units in 1 hour, or 1 unit in 10 hr

(if enzyme stable that long – many are not)

ideal reaction conditions depend on enzyme

– different enzymes have different requirements

– salt concentration

– wrong concentration may cause lower activity

– or “star” activity

– altered cleavage specificity

– temperature – 37ºC ideal for most

– 50-65ºC better for thermophile enzymes

– type of DNA

– supercoiled plasmid DNA

– cleaved at lower efficiency

– may need more enzyme/time to cut

– source of DNA

– methylation of RE site

– may interfere with cleavage

RE buffers: storage & activity buffers

– storage buffers maximise stability of enzyme

(in stock solution)

– contain high concentrations glycerol

– interferes with enzyme activity

– enzyme must be </= to ¹/₁₀ total reaction volume

– activity buffers maximise activity – not enzyme stability

– used only during DNA digests

– supplied with enzyme

partial digests – use less enzyme (or shorter time)

– cleave some of RE sites for enzyme

– random choice among sites