E.

coli DNA Polymerase I

The E. coli DNA polymerase I is a DNA-dependent DNA polymerase that possesses both 3'
-> 5' and 5' -> 3' exonclease acti!ities. It is a single-chain protein with a mass of about
109,000 Da that requires magnesium as a cofactor. Each of its three enzymatic actiities are
encapsulate! into !istinct !omains of the holoenzyme, such that proteolytic !eletions can be
generate! that lac" one or more of the actiities. #he so-calle! $lenow fragment is one such
molecule that is wi!ely use! in recombinant D%& wor".
D%& polymerase I was use! frequently in the early !ays of recombinant D%& technology for
ra!iolabeling D%& an! synthesizing cD%&. 'oweer, other enzymes hae proen to be more
effectie for these purposes, inclu!ing a proteolytic fragment of D%& polymerase I calle!
$lenow fragment an! #( D%& polymerase. #he holoenzyme D%& polymerase I is no longer
frequently use!.
"ar#e $%leno&' (ra#ment o) E. coli
DNA Polymerase I
#he )* -+ ,* e-onuclease actiity of E. coli*s D%& polymerase I ma"es it unsuitable for
many applications. 'oweer, this pes"y enzymatic actiity can rea!ily be remoe! from
the holoenzyme. E-posure of D%& polymerase I to the protease subtilisin cleaes the
molecule into a small fragment, which retains the )* -+ ,* e-onuclease actiity, an! a
large piece calle! $lenow fragment. The lar#e or %leno& )ra#ment o) DNA
polymerase I has DNA polymerase and 3' -> 5' exonclease acti!ities* and is &idely
sed in moleclar biolo#y.
In a!!ition to generating $lenow fragment by proteolysis, it can be e-presse! in bacteria
from a truncate! form of the D%& polymerase I gene. #he enzyme you purchase is
almost certainly pro!uce! in this manner.
%leno& )ra#ment is se)l )or se!eral tas+s,
-ynthesis o) doble-stranded DNA )rom sin#le-stranded templates, #he function of
D%& polymerases is to synthesize complementary stran!s !uring D%& replication.
.erforming that tas" in the lab is integral to such processes as synthesizing the secon!
stran! D%& in cD%& cloning an! generating ra!ioactie probes for hybri!ization
reactions.
D%& polymerases require a primer to proi!e a free ,* hy!ro-yl group for initiation of
synthesis. #he primers use! for most in itro polymerization reactions are single-
stran!e! D%&s, typically / to 00 bases in length, calle! oligonucleoti!es. #he
oligonucleoti!es must be complemenary to some section of template D%&. #o use
$lenow to synthesize a complementary stran! of D%&, one simply mi-es single-
stran!e! template 1usually !enature! !ouble-stan!e! D%&2, primers an! the enzyme in
the presence of an appropriate buffer 1most restriction enzyme buffers wor" well2. #he
reaction procee!s are !epicte! below3
4ne item of some significance in the aboe reaction is that as $lenow procee!s, it can
!isplace primers !ownstream an! continue synthesizing new D%&.
(illin# in recessed 3' ends o) DNA )ra#ments, & 5fill-in5 reaction is use! to create
blunt en!s on fragments create! by cleaage with restriction enzymes that leae )*
oerhangs. #his reaction is conceptually i!entical to the one !escribe! aboe, but with a
huge primer an! a ery short segment of single-stran!e! template.
Di#estin# a&ay protrdin# 3' o!erhan#s, #his is another metho! for pro!ucing blunt
en!s on D%&, in this with en!s generate! from restriction enzymes that cleae to
pro!uce ,* oerhangs. #he ,* -+ )* e-onuclease actiity of $lenow will !igest away the
protru!ing oerhang. 6emoal of nucleoti!es from the ,* en!s will continue, but, in the
presence of nucleoti!es, the polymerase actiity will balance the e-onuclease actiity,
yiel!ing blunt en!s. #his reaction is more efficienty con!ucte! with #( D%&
polymerase, which has much more potent e-onuclease actiity.
Preparation o) radioacti!e DNA probes, E-amine each of the reactions !epicte!
aboe. What if the nucleotides used were in the reaction were radioactive? #hat*s correct
- the ra!ioactie nucleti!es woul! be incorporate! into the D%& fragment. $lenow
fragment is use! frequently to prepare D%& that is labele! with ra!ionucli!es or other
mar"ers.
In some sitations* the 3' -> 5' exonclease acti!ity o) %leno& )ra#ment is either
ndesirable or not necessary. 7y intro!ucing mutations in the gene that enco!es
$lenow, forms of the enzyme can be e-presse! that retain polymerase actiity, but lac"
any e-onuclease actiity. #hese forms are the enzyme are usually calle! e-o
-
$lenow
fragment.
T. DNA Polymerase
#( is a bacteriophage of E. coli. The acti!ities o) T. DNA polymerase are !ery similar
to %leno& )ra#ment of D%& polymerase I - it functions as a )* -+ ,* D%& polymerase
an! a ,* -+ )* e-onuclease, but !oes not hae )* -+ ,* e-onuclease actiity.
In general, #( D%& polymerase is use! for the same types of reactions as $lenow
fragment, particularly in blunting the en!s of D%& with )* or ,* oerhangs. There are
however, two differences between the two enzymes that have practical signficance:
#he ,* -+ )* e-onuclease actiity of #( D%& polymerase is roughly 000 times
that of $lenow fragment, ma"ing it preferre! by many inestigators for blunting
D%&s with ,* oerhangs.
8hile $lenow fragment will !isplace !ownstream oligonucleoti!es as it
polymerizes, #( D%& polymerase will not. #his attribute ma"es #( D%&
polymerase the more efficient choice for certain types of oligonucleoti!e
mutagenesis reactions.
T/ DNA Polymerase
The DNA polymerase o) T/ bacteriopha#e has DNA polymerase and 3' -> 5'
exonclease acti!ities* bt lac+s a 5' -> 3' exonclease domain. It is thus ery similar
in actiity to $lenow fragment an! #( D%& polymerase.
The claim to )ame )or T/ DNA polymerase is it's processi!ity. #hat is to say, the
aerage length of D%& synthesize! before the enzyme !issociates from the template is
consi!erably greater than for other enzymes. Due to this talent, the principle se o) T/
DNA polymerase is in DNA se0encin# by the chain termination techni0e.
#9 D%& polymerase can be chemically-treate! or genetically engineere! to abolish it*s ,*
-+ )* e-onuclease actiity. #hese forms of the enzyme are mar"ete! un!er the name
-e0enase and -e0enase 1.2, an! are wi!ely use! for D%& sequencing reactions.
Terminal Trans)erase
Terminal trans)erase cataly3es the addition o) ncleotides to the 3' termins o)
DNA. Interestin#ly* it &or+s on sin#le-stranded DNA* incldin# 3' o!erhan#s o)
doble-stranded DNA* and is ths an example o) a DNA polymerase that does not
re0ire a primer. It can also a!! homopolymers of ribonucleoti!es to the ,* en! of
D%&.
#he much preferre! substrate for this enzyme is protru!ing ,* en!s, but it will also, less
efficiently, a!! nucleoti!es to blunt an! ,*-recesse! en!s of D%& fragments. :obalt is a
necessary cofactor for actiity of this enzyme.
Terminal trans)erase is se)l )or at least t&o procedres,
"abelin# the 3' ends o) DNA, ;ost commonly, the substrate for this reaction is a
fragment of D%& generate! by !igestion with a restriction enzyme that leaes a ,*
oerhang, but oligo!eo-ynucleoti!es can also be use!. 8hen such D%& is incubate!
with tagge! nucleoti!es an! terminal transferase, a string of the tagge! nucleoti!es will
be a!!e! to the ,* oerhang or to the ,* en! of the oligonucleoti!e.
Addin# complementary homopolymeric tails to DNA, #his cleer proce!ure was
commonly use! in the past to clone cD%&s into plasmi! ectors, but has largely been
replace! by other, much more efficient techniques. #he principles of this technique are
!epicte! in the figure below. 7asically, terminal transferase is use! to tail a linearize!
plasmi! ector with <*s an! the cD%& with :*s. 8hen incubate! together, the
compementary <*s an! :*s anneal to 5insert5 the cD%& into the ector, which is then
transforme! into E. coli.
Terminal trans)erase is a mammalian en3yme* expressed in lymphocytes. #he
enzyme purchase! commercially is usually pro!uce! by e-pression of the boine gene in
E. coli.
Thermostable DNA Polymerases
It is interesting how some seemingly esoteric or obscure
!iscoeries can, years later, be catapulte! to something of
immense practical importance. =uch is the history of #aq D%&
polymerase. #he original report of this enzyme, purifie! from the
hot springs bacterium Thermus aquaticus, was publishe! in 199/.
6oughly 10 years later, the polymerase chain reaction was
!eelope! an! shortly thereafter 5#aq5 became a househol! wor!
in molecular biology circles. :urrently, the worl! mar"et for #aq polymerase is in the
hun!re!s of millions of !ollars each year.
The thermophilic DNA polymerases* li+e other DNA polymerases* cataly3e
template-directed synthesis o) DNA )rom ncleotide triphosphates. & primer haing
a free ,* hy!ro-yl is require! to initiate synthesis an! magnesium ion is necessary. In
general, they hae ma-imal catalytic actiity at 9) to >0:, an! substantially re!uce!
actiites at lower temperatures. &t ,9:, #aq polymerase has only about 10? of its
ma-imal actiity.
In a!!ition to #aq D%& polymerase, seeral other thermostable D%& polymerases hae
been isolate! an! e-presse! from clone! genes. #hree of the most-use! polymerases are
!escribe! in the following table3
Polymerase
3'->5'
Exonclease
-orce and Properties
Ta0 %o @rom Thermus aquaticus. 'alflife at 9): is 1./ hours.
P) Aes
@rom Pyrococcus furiosus. &ppears to hae the lowest
error rate of "nown thermophilic D%& polymerases.
4ent Aes
@rom Thermococcus litoralisB also "nown as #li
polymerase. 'alflife at 9) : is appro-imately 9 hours.
In a!!ition to the natie polymerases liste! in the table aboe, a number of mutants hae
been generate! an! are aailable, for e-ample, a form of Cent polymerase that lac"s the
,*-+)* e-onuclease an! is thereby more similar to #aq.
5ne o) the most discssed characteristics o) thermostable polymerases is their
error rate. Error rates are measure! using seeral !ifferent assays, an! as a result,
estimates of error rate ary, particularly when the assays are performe! by !ifferent labs.
&s woul! be e-pecte! from first principles, polymerases lac+in# 3'->5' exonclease
acti!ity #enerally ha!e hi#her error rates than the polymerases &ith exonclease
acti!ity. #he total error rate of #aq polymerase has been ariously reporte! between 1 -
10
-(
to 0 - 10
-)
errors per base pair. .fu polymerase appears to hae the lowest error rate
at roughly 1.) - 10
-/
error per base pair, an! Cent is probably interme!iate between #aq
an! .fu.
Error rate is not the only consideration in chosin# a polymerase )or P67, otherwise
#aq polymerase woul! not continue to be the most wi!ely use! enzyme by far for the
myria! of .:6 applications. 4ther consi!erations, inclu!ing reliability an! what might
be calle! 5fussiness5 enter into the choice, as !iscusse! further in the section on
.olymerase :hain 6eaction #echnology.
7e!erse Transcriptases
7e!erse transcriptase is a common name )or an en3yme that
)nctions as a 7NA-dependent DNA polymerase. #hey are enco!e!
by retroiruses, where they copy the iral 6%& genome into D%&
prior to its integration into host cells.
7e!erse transcriptases ha!e t&o acti!ities,
DNA polymerase acti!ity, In the retroiral life cycle, reerse transcriptase
copies only 6%&, but, as use! in the laboratory, it will transcribe both single-
stran!e! 6%& an! single-stran!e! D%& templates with essentially equialent
efficiency. In both cases, an 6%& or D%& primer is require! to initiate synthesis.
7Nase 8 acti!ity, 6%ase ' is a ribonuclease that !egra!es the 6%& from 6%&-
D%& hybri!s, such as are forme! !uring reerse transcription of an 6%&
template. #his enzyme functions as both an en!onuclease an! e-onuclease in
hy!rolyzing its target.
All retro!irses ha!e a re!erse transcriptase* bt the en3ymes that are a!ailable
commercially are deri!ed )rom one o) t&o retro!irses, either by purification from
the irus or e-pression in E. coli3
9oloney mrine le+emia !irs3 a single polypepti!e
A!ian myeloblastosis !irs3 compose! of two pepti!e chains
7oth enzymes hae the same fun!amental actiities, but !iffer in a number of
characteristics, inclu!ing temperature an! p' optima. ;ost importantly, the murine
leu"emia irus enzyme has ery wea" 6%ase ' actiity compare! to the aian
myeloblastosis enzyme, which ma"es it the clear choice when trying to synthesize
complementary D%&s for long messenger 6%&s.
7e!erse transcriptase is sed* as yo mi#ht
expect* to copy 7NA into DNA. #his tas" is
integral to cloning complementary D%&s
1cD%&s2, which are D%& copies of mature
messenger 6%&s. :loning cD%&s is !iscusse!
elsewhere in more !epth, but the technique is
usually initiate! by mi-ing short 110-1> base2
polymers of thymi!ine 1oligo !#2 with
messenger 6%& such that they anneal to the 6%&*s polya!enylate tail. 6eerse
transcriptase is then a!!e! an! uses the oligo !# as a primer to synthesize so-calle! first-
stran! cD%&.
Another common se )or re!erse transcriptase is to #enerate DNA copies o) 7NAs
prior to ampli)yin# that DNA by polymerase chain reaction $P67'. 6eerse
transcription .:6, usually calle! simply 6#.:6, is a stupifyingly useful tool for such
things as cloning cD%&s, !iagnosing microbial !iseases rapi!ly an! a myria! of other
applications. In most cases, stan!ar! preparations of reerse transcriptase are use! for
6#.:6, but mutate! forms with relatiely high thermal stability hae been !eelope! to
facilitate the process.
:acteriopha#e 7NA Polymerases
Pha#e-encoded DNA-dependent 7NA polymerases are sed )or in !itro
transcription to #enerate de)ined 7NAs. ;ost commonly, the reaction utilizes
ribonucleoti!es that are labele! with ra!ionucli!es or some other tag, an! the resulting
labele! 6%& is use! as a probe for hybri!ization. 4ther applications of in itro
transcription inclu!ing ma"ing 6%&s for in itro translation or to stu!y 6%& struction
an! function.
-e!eral bacteriopha#e 7NA polymerases are commercially a!ailable. #hey are
name! after the phage that enco!es them, an! either purifie! from phage-infecte!
bacteria or pro!uce! as recombinant proteins3
Polymerase Name
8ost o) Encodin#
Pha#e
Promoter -e0ence
T/ 7NA
polymerase
E coli #&&#&:<&:#:&:#&#&<<<
T3 7NA
polymerase
E coli &&##&&:::#:&:#&&&<<<
-P; 7NA
polymerase
!almonella
typhimurium
&&###&<<#<&:&:#&#&<&&
9any o) the plasmids sed )or carryin# cloned DNA incorporate promoters )or
bacteriopha#e 7NA polymerases ad<acent to the clonin# site. #his allows one to
rea!ily obtain either m6%&-sense or antisense transcripts from the inserte! D%&. #he
process is often calle! run-off transcription, because the plasmi! is cut with a restriction
enzyme !ownstream of the inserte! D%&, which causes the polymerase to fall off the
template when it reaches that spot.
If we assume that the 6%& transcribe! in the figure aboe has the polarity of a m6%&
1e.g. sense2, it is easy to mo!ify the construct to e-press an antisense 6%& - simply
reerse the orientation of the transcribe! region. In!ee!, most plasmi!s use! for in itro
transcription hae two !ifferent phage polymerase promoters flan"ing the insertion site,
which allows transcription of sense 6%& with one polymerase an! antisense with the
other.
Ncleases, DNase and 7Nase
9ost o) the time ncleases are the enemy o) the moleclar biolo#ist &ho is tryin# to
preser!e the inte#rity o) 7NA or DNA samples. 8o&e!er* deoxyriboncleases
$DNases' and riboncleases $7Nases' ha!e certain indispensible roles in moleclar
biolo#y laboratories.
%umerous types of D%ase an! 6%ase hae been isolate! an! characterize!. #hey !iffer
among other things in substrate specificity, cofactor requirements, an! whether they
cleae nucleic aci!s internally 1en!onucleases2, chew in from the en!s 1e-onucleases2 or
attac" in both of these mo!es. In many cases* the sbstrate speci)icity o) a nclease
depends pon the concentration o) en3yme sed in the reaction, with high
concentrations promoting less specific cleaages.
The most &idely sed ncleases are DNase I and 7Nase A, both of which are purifie!
from boine pancreas3
Deoxyribonclease I cleaes !ouble-stran!e! or single stran!e! D%&. :leaage
preferentially occurs a!Dacent to pyrimi!ine 1: or #2 resi!ues, an! the enzyme is
therefore an en!onuclease. ;aDor pro!ucts are )*-phosphorylate! !i, tri an!
tetranucleoti!es.
In the presence of magnesium ions, D%ase I hy!rolyzes each stran! of !uple- D%&
in!epen!ently, generating ran!om cleaages. In the presence of manganese ions, the
enzyme cleaes both stran!s of D%& at appro-imately the same site, pro!ucing blunt
en!s or fragments with 1-0 base oerhangs. D%ase I !oes not cleae 6%&, but cru!e
preparations of the enzyme are contaminate! with 6%ase &B 6%ase-free D%ase I is
rea!ily aailable.
=ome of the common applications of D%ase I are3
Eliminating D%& 1e.g. plasmi!2 from preparations of 6%&.
&nalyzing D%&-protein interactions ia D%ase footprinting.
%ic"ing D%& prior to ra!iolabeling by nic" translation.
7ibonclease A is an en!oribonuclease that cleaes single-stran!e! 6%& at the ,* en! of
pyrimi!ine resi!ues. It !egra!es the 6%& into ,*-phosphorylate! mononucleoti!es an!
oligonucleoti!es.
=ome of the maDor use of 6%ase & are3
Eliminating or re!ucing 6%& contamination in preparations of plasmi!
D%&.
;apping mutations in D%& or 6%& by mismatch cleaage. 6%ase will
cleae the 6%& in 6%&3D%& hybri!s at sites of single base mismatches,
an! the cleaage pro!ucts can be analyze!.
& number of other nucleases that are use! to manipulate D%& an! 6%& are !escribe! in
the following table3
Nclease and
-orce
-bstrates* Acti!ity and =ses
Exonclease
III
1E. coli2
6emoes mononucleoti!es from the ,* termini of !uple- D%&. #he
preferre! substrates are D%&s with blunt or )* protru!ing en!s. It will
also e-ten! nic"s in !uple- D%& to create single-stran!e! gaps. In
wor"s inefficiently on D%& with ,* protru!ing en!s, an! is inactie on
single-stran!e! D%&.
Ese! most commonly to prepare a set of neste! !eletions of the
termini of linear D%& fragments.
9n# :ean
Nclease
1;ung bean
sprouts2
Digests single-stran!e! D%& to )*-phosphorylate! mono or
oligonucleoti!es. 'igh concentrations of enzyme will also !egra!e
!ouble-stran!e! nucleic aci!s.
Ese! to remoe single-stran!e! e-tensions from D%& to pro!uce
blunt en!s.
Nclease :A"
3>
1"lteromonas2
@unctions as an e-onuclease to !igest both )* an! ,* en!s of !ouble-
stran!e! D%&. It also acts as a single-stran!e! en!onuclease that
cleaes D%& at nic"s, gaps an! single stran!e! regions. Does not
cleae internally in !uple- D%&.
Ese! for shortening fragments of D%& at both en!s.
Nclease ->
1"spergillus2
#he substrate !epen!s on the amount of enzyme use!. Fow
concentrations of =1 nuclease !igests single-stran!e! D%&s or 6%&s,
while !ouble-stran!e! nucleic aci!s 1D%&3D%&, D%&36%& an!
6%&36%&2 are !egra!e! by large concentrations of enzyme.
;o!erate concentrations can be use! to !igest !ouble-stran!e! D%&
at nic"s or small gaps.
Ese! commonly to analyze the structure of D%&36%& hybri!s 1=1
nuclease mapping2, an! to remoe single-stran!e! e-tensions from
D%& to pro!uce blunt en!s.
7ibonclease
T>
1"spergillus2
&n en!onuclease that cleaes 6%& at ,* phosphates of guanine
resi!ues, pro!ucing oligonucleoti!es terminal guanosine ,* phosphates.
Ese! to remoe unanneale! regions of 6%& from D%&36%& hybri!s.
DNA "i#ase
DNA li#ases cataly3e )ormation o) a phosphodiester bond bet&een the 5' phosphate
o) one strand o) DNA and the 3' hydroxyl o) the another. #his enzyme is use! to
coalently lin" or ligate fragments of D%& together. ;ost commonly, the reaction
inoles ligating a fragment of D%& into a plasmi! ector, which is a fun!amental
technique in recombinant D%& wor".
#he most wi!ely use! D%& ligase is !erie! from the #( bacteriophage. #( D%& ligase
requires &#. as a cofactor. & D%& ligase from E. coli is also aailable, but is not
commonly use!. #he E. coli enzyme uses %&D as a cofactor.
&!!itional information on the use of #( D%& ligase is presente! in the section on D%&
ligation.
Al+aline Phosphatase
Al+aline phosphatase remo!es 5' phosphate #rops )rom DNA and 7NA. It will also
remoe phosphates from nucleoti!es an! proteins. #hese enzymes are most actie at
al"aline p' - hence the name.
There are se!eral sorces o) al+aline phosphatase that di))er in ho& easily they can
be inacti!ated,
:acterial al+aline phosphatase $:AP' is the most actie of the enzymes, but
also the most !ifficult to !estroy at the en! of the !ephosphorylation reaction.
6al) intestinal al+aline phosphatase $6IP' is purifie! from boine intestine.
#his is phosphatase most wi!ely use! in molecular biology labs because,
although less actie than 7&., it can be effectiely !estroye! by protease
!igestion or heat 19): for 10 minutes in the presence of ) m; ED#&2.
-hrimp al+aline phosphatase is !erie! from a col!-water shrimp an! is
promote! for being rea!ily !estroye! by heat 1/): for 1) minutes2.
There are t&o primary ses )or al+aline phosphatase in DNA maniplations,
7emo!in# 5' phosphates )rom plasmid and bacteriopha#e !ectors that hae
been cut with a restriction enzyme. In subsequent ligation reactions, this
treatment preents self-ligation of the ector an! thereby greatly facilitates
ligation of other D%& fragments into the ector 1e.g. subcloning2.
7emo!in# 5' phosphates )rom )ra#ments o) DNA prior to labelin# &ith
radioacti!e phosphate. .olynucleoti!e "inase is much more effectie in
phosphorylating D%& if the )* phosphate has preiously been remoe!.
It is usually recommen!e! that !ephosphorylation of D%&s with blunt or )*-recesse!
en!s be con!ucte! using a higher concentration al"aline phosphatase or at higher
temperatures than for D%&s with )* oerhangs.
Polyncleotide %inase
Polyncleotide +inase $PN%' is an en3yme that cataly3es the trans)er o) a phosphate
)rom ATP to the 5' end o) either DNA or 7NA. It is a pro!uct of the #( bacteriophage,
an! commercial preparations are usually pro!ucts of the clone! phage gene e-presse! in
E. coli.
The en3ymatic acti!ity o) PN% is tili3ed in t&o types o) reactions,
In the ?)or&ard reaction?, .%$ transfers the gamma phosphate from &#. to
the )* en! of a polynucleoti!e 1D%& or 6%&2. #he target nucleoti!e is lac"ing a
)* phosphate either because it has been !ephorphorylate! or has been synthesize!
chemically.
In the ?exchan#e reaction?, target D%& or 6%& that has a )* phosphate is
incubate! with an e-cess of &D. - in this setting, .%$ will first transfer the
phosphate from the nucleic aci! onto an &D., forming &#. an! leaing a
!ephosphorylate! target. .%$ will then perform a forwar! reaction an! transfer a
phosphate from &#. onto the target nucleic aci!.
&s you might e-pect, the efficiency of phosphorylating ia the e-change reaction is
consi!erably less than for the forwar! reaction. In a!!ition to its phosphorylating
actiity, .%$ also has ,* phosphatase actiity, although this has little significance to
molecular tecnologists.
There are t&o ma<or indications )or phosphorylatin# ncleic acids and hence se o)
PN% are,
.hosphorylating lin"ers an! a!aptors, or fragments of D%& as a prelu!e to
ligation, which requires a )* phosphate. #his inclu!es pro!ucts of polymerase
chain reaction, which are typically generate! using non-phosphorylate! primers.
6a!iolabeling oligonucleoti!es, usually with
,0
., for use as hybri!ization probes.
.%$ is inhibite! by small amounts of ammonium ions, so ammonium acetate shoul! not
be use! to precipitate nucleic aci!s prior to phosphorylation. Fow conceptrations of
phosphate ions, or %a:l concentrations greater than about )0 m;, also inhibit this
enzyme.