Ligation

– joining DNA fragments together

– formation of covalent phosphodiester link

– by ligase enzyme

– most commonly used ligase: T4 DNA ligase

– from bacteriophage T4 (gene now cloned in E. coli)

– uses ATP cofactor

– best results – 16°C overnight

– balance between:

– kinetic energy

(lower temperature, less molecular motion)

– enzyme activity (increases up to 30°C)

– enzyme unstable at higher temperatures

– decent results (~ 50% of maximum)

– 30 minutes room temperature

– activity increased by crowding reagents

– e.g. polyethylene glycol (PEG)

– bulky macromolecules

– crowd DNA ends together

– increase intermolecular ligation, not intramolecular

– 2 ligations needed for plasmid insert

– 1^st is intermolecular – plasmid to insert

– second is intramolecular

– joining other ends

– 1^st reaction increased by PEG, 2^nd decreased

– may cause linear concatemers to form

– may join:

matching cohesive ends

– must have same 5' or 3' extension

– efficient reaction – 2 stages

– annealing of ends

– based on complimentary H-bonding

– joining of nicks by ligase

blunt ends – always “match” – all the same

– no annealing possible

– 2 ends must ligate when bump into each other

– lower efficiency of ligation

– need higher concentrations of enzyme

– other ligases:

Escherichia coli DNA ligase – E. coli enzyme

– uses NAD⁺ as cofactor

– ligates cohesive ends well

– ligates blunt ends with very low efficiency

– greater specificity than T4 enzyme

– e.g. T4 ligase ligates DNA/RNA hybrids

(cDNA second strand)

– E. coli enzyme will not – only DNA strand

Thermophile ligases – e.g. Taq ligase

– use NAD⁺ as cofactor

– ligates blunt ends with very low efficiency

– heat stable

problems with ligation (& solutions)

– vector self-ligation – use alkaline phosphatase

– removes 5' phosphate from vector

– vector only ligates to insert (has 5' phosphate)

problems

– lower rates of ligation with phosphatased vectors

– must inactivate phosphatase before ligation set up

– or insert dephosphorylated as well

calf intestinal phosphatase (CIP)

– inactivate with SDS, EDTA, proteinase, heat

– preferably all at once

– then phenol/chloroform extract

– losses of DNA a problem (small volumes)

– unless dilute sample/re-extract interface

shrimp alkaline phosphatase (SAP)

– from arctic shrimp species – heat sensitive

– inactivate 15 minutes, 65°C (in theory)

cohesive end ligations – simple (if ends match)

– can do directional cloning

– cut vector (& insert) with 2 enzymes

– ends of each fragment do not match

– will not self-ligate

– no phosphatase needed

What if ends do not match?

– can convert both ends to blunt ends

– lower efficiency ligation

5' extensions (3' recessed ends)

– usually end-filled with Klenow + dNTPs

3' extensions (5' recessed ends)

– T4 DNA polymerase used

– strong 3'-5' exonuclease activity

– can also remove 3' or 5' single-stranded extensions

– using single stranded exonucleases

(S1 nuclease, mung bean nuclease)

– digest single-stranded DNA, not double-stranded

– but products do not ligate well

mismatched ends sometimes converted to matched ends

– by partial end-filling

e.g. XhoI – end-fill with dTTP, dCTP

C TCGAG

GAGCT C

& Sau3A – end-fill with dGTP, dATP

N GATCN

NCTAG N

– ends of fragments cannot self-ligate

– but can ligate to other filled end

avoiding blunt end ligation

– for blunt-cutter RE, filled ends, cDNAs, etc.

– inefficient

– ligated blunt ends from different enzymes

– cannot (usually) be recut

– to remove cloned fragment

(unless sequences line up to make site)

linkers

– pair of homologous oligonucleotides

– RE site in middle, extra sequence on ends

(most REs do not cut at end of molecule)

– hybridize together – double stranded sequence

(can be cut by RE) (Fig. 2.10)

– ligate linkers to blunt ends

(high concentrations of linker – blunt ligation)

– cut with RE – linker concatemer removed

– left with cohesive end on fragment

problem – what if same RE site in fragment as linker?

– cleavage will remove part of fragment

– solve with methylase treatment

– type II REs have matching methylase enzymes

– protect host DNA

– some methylases cloned – available commercially

– treat blunt fragment with methylase for specific site

– sites in fragment protected – inactivate methylase

– ligate linkers to end – digest & only cut linkers

adaptors (Fig. 2-12)

– similar to linkers, but have 1 or 2 cohesive ends

– can ligate blunt/cohesive onto blunt

– cohesive end without extra digest

– can ligate cohesive/cohesive onto first end

– change ends to different site

– 1 end of adaptor usually not phosphorylated

– no concatemers form

– and no need to phosphatase

(if PO₄ needed, can add later with polynucleotide kinase)

extraction of DNA from agarose gels

electrophoretic techniques

– elute DNA from gel onto membrane using electrophoresis

– or into dialysis bags

– very effective & gentle (large fragments) – but tedious

low melting point agarose – modified agarose

– melts at 65°C

– remove polysaccharides with phenol extraction

– expensive & not good for large fragments

agarase enzyme – digests agarose

– gentler technique, but inconsistent results

sodium iodide – dissolves agarose

– purify DNA using silica matrix – like miniprep

– many kits available

– simple, (usually) good results

– centrifugation may shear large fragments

introduction of plasmid DNA into E. coli cells

– 2 main techniques:

chemical transformation

– cells made “competent” to take up DNA

– cold temperatures, divalent cations (Ca²⁺, Mg²⁺)

– mechanism not completely understood

– may involve destabilizing Gram negative cell wall

– much higher transformation rates for circular molecules

– e.g. closed circular plasmid ligation products

– linear concatemers not taken up by cells

electroporation

– transient gaps in cell membrane induced by electric shock

– at 0°C, gaps stay open long enough for DNA to enter cell

– highly effective technique

– high yields of transformed cells

– expensive equipment