A Rapid Method for
Creating Recombinant
DNA Molecules
Ttrc construction of recombinant
DNA moleculesreprsents a molecular
versionof aclassicalgencic cross,and
is a basictechniqueof molequlargenetics. At the request of the editors, I
describea rapid mchod for in vitro
,:onstruction of recombinant DNA
rnoler,-r.rles
that my laboratory hasused
'ior the past 5 years (see Gene
i5:].31-241, 1983).The "method"
reprcsenca coUectionof tricks whose
original sourcesare obscure(at leastto
rne). The procedureis as follows:
Plasmid or phage DNAs (rf'pically
tl.l to 2"A ygl, preparedby essentially
any rapid lysateprocedure,are treated
with appropriate enzymesand the r+
:;uhing prod uctsare eloctrophoroically
separatedon low gelling/melting temperarureagarosein a buffer of 50 mM
'I'ris-acetae,
pH 8.2. Agaroseconcent,:ationsrangrng from 0.5 to 2u/ohave
hen usedsuccessfully.The sourceof
the agarcseis critical; SeaPlaqueQproducedby Marine Colloids (Rockland,
ME) has always been reliable. The
Cesiral DNA .segrnents,
visualizedby
iong-waveultraviolc tight after staining with sthidium bromide, areexcised
i'::ornrhegel with a cleanrazorbladein
a.;mrall a volume aspoesible(ustrally3O
tc 50yI). Cel slicescontainingthe relevart DNA seglnents
aremeltedat 70t
for 5 to l5 minutesand thcncombined
in ;rypropriate proponions to give a
final vnlumeof l0 Fl. After equilibratrcn *i'the moltengelslicesto 37oC,l0
pl oi"ice-cold,2 x concentratdbuffer
conteiningT4 or T7 DNA tigaseisadded, rnired quickly, and the mixture is
thcn irrcubatedat lsqc for 3 to 2A
hours. Although the reaction mixture
resolidifiesinto agd, the lipti'cn works;
indeed, the reaction is barely, if at dl,
inhibited by the agaroee!To inuoduce
tlp ligatedproductsinto E coltodls, thc
gd containingthe reactionmixnue is re
meltedat 70qCand diluted by a factor
of t0 to.50into ice<old TCM (t0 mM
Tris, pH 7.5, l0 mM MgCl2, l0 mM
CaCl') prior to carrying out the standard transformation procedure. AIthough not explicitly examined, it is
likely that the dilution steppreventsr+
gelling of the agarose.We have never
testedwhetherligatedproducts within
the gefmatrix can be packagedin vitro
into viableI phages.
Severaltechnical points are worth
noting. First, almostall of theenzym€s
usedfor DNA cloning areactivein molten or resolidifiedSeaPlaquePagarose.
This includesessentiallyall restriaion
endonucleases,DNA ligases, DNA
polymeraseI (for end-Frllingand nicktranslation reactions), BAL-31 nuclease, and calf intestinal alkaline
phosphatase.Obviously, SeaPlaqu€p
is free of the inhibitory components
found in most commercialpreparations
of agarose;presumablythis reflectsa
higherdegreeof purification. The only
enzymethat appearsto be inhibited reproducibly by the low gelling/melting
temperatureagaros€is T4 polynucl+
tide kinase.Second,very little DNA is
necessary;basically, if a band can be
visualized,the hybrid constructionis
very likely to be successful.lndeed, a
singlegel slicecan be usedfor 5 to 20
s€parateligationreactions,and it canbe
storedand repeatedlyremeltedfor use.
'[hird,
the concentrations
of the inpuf
DNA fragrnenrs;uegenerallyof minlmal importanceand can be very low.
The major exceptionto this ruleoccurs
if one or more of the DNAScan circuVol.3, No. 6 ( l9t5)
larizeto producea selectable,ransforming moleculecapableof autonomoull
replication; without specialmanipulations, the backgroundof "parental"
moleculeswill be extremelyhieh. Thi$
problemis easilyavoidedby usinginput
DNAS cleavedwith 2 different restriction enzymesor by treatment*ith cal{'
intestinalalkaline phosphatase.Fourth,
the methodworks for complicatedconstmctions involving 3-fragment ligations, DNA fragments produced by
partid cleavagewith restriction enzymes, blunt-endedligations, ancl
- BAL-31 delaion mutants. Fifth, the
electrophoreticsdparation removes
linkers(whichoften in-'
oligonucleotide
terfere with subsequentligation reactions), as well as the enzymesusecltr:
cleaveor modify the DNA (thuselim!nating the needto destroysuchenzyrne;
by phenolextractionand/or heatinactivation).
The methodhasa numbet'of advantages.The eletrophoretic purification
of DNA fraggnents(includingthe cloning vector) minimizes the problerm
causedby incomplete digestion by
and rnakesit
restric"tionendonucleas€s
possibleto start with crud€ prr:p:uations. Moreover, as only the desireci
DNA segnentsare includd in the ligation reactionmixture, the backgrouncl
of undesiredmoleculesis greatlyreduoed.
which is parttularly important becaus:
analysisof the transformants is often
the rate-limitingstepin a construction.
For simpleconstructions,the ntajclnt-r
of tlrc transformantscontain the desired
DNA molecule,and for morecomPlex
situationsthe trequencyis sufficiently
high to avoid time consumingscteening
proceduressuchasfilter hybridization"
Although numerousother methodsare
availablefor purifying DNA segrnents
from agaros€geh, the major advantage
of the proceduredccribed hereis that
it is both considerablyfasterand more
reliable.An experimentalistdedicated
to speedcanoften achievea molecular
generationtime of 2-3 days.I
Kevin Struhl
Department of Biological
ChembtrY
Harvard Medical School
Bostan, MA 021I5
Vol. 3.No. 6 (lgEt)