Note: Descriptions are shown in the official language in which they were submitted.
WO93/19~6 PCT/~S93/0~422
CA 21 ~ 7 582
SYNT~ESIS OF FLUORESCENCE-L~BELLED NUCLEIC ACIDS
BACKGROUND OF ~HE I~VENTION
~:: This is a continu~tion-in-part of application Serial No.
:~507/852,705, fil d ~arch 17, 1992. The field of the invention is
: enzyme-catalyzed DNA synthesis particularly synthesis of
fluorescence-labell~d DNA. ~srP particularly, the invPntion
relates to synthesi~ of fluoresc~nce-labelled nuGleic acids
h~ving: sequeDce complementary to a specified ~ucleic acid
~:~ 10 template.
echniques for labelling nucleic aGids with a flu~res~ence
marker can~ be divided: i~to two categories: pos -~ynthetic
chemical or enzymatic modification, and incorporation of
fluorescence-labelled precursors during ~ynthesis. For examples
~of the former category~see Dirks, R.W. et al~ (l991) Exptl. Cell
~,
Res. 194:310-315;: Lichter, P. et al. (1988~ Human Genetics
80:224-234; and Li hter, P. et al. ~1990) Proc. Natl. ~cad. Sci.
USA 87:6634-6638. The present invention belo~gs in the latter
category. A typical enzyme-catalyzed DNA synthesis reaction
employs a template DNA or RNA, a primer oligonucl otide having
.~a sequence c~mplementary to a segment of the tempIata DNA, an
enzyme catalyst and ~our deoxynucleotide (dNTP) precursors, dATP,
dGTP, dCTP and dTTP or, alternatively, dUTP. Varîous enzyme
catalysts are known in the art, such as E. coli DNA polymerase,
T7 ~NA polymerase, Klenow fragment of DNA polymerase, Taq DNA
,
WO93/19206 C A 2 i i 7 5 82 PCT/US93/02422
polymerase, reverse transcriptase and the like. The enzymes c~n
be characterized ~y the degree to which an enzyme molecule
remains continuously associated with the same growing strand of
nascent DNA. Tho~e enzymes which tend to remain associated with
the same nascent DNA strand for longer times are termed
"processive." In general, the term "prscessivity" refers to the
number of nucleotides that a D~A polymerase incorporates during
DNA synthesis be~ore dis~ociating from the template-primer
complex. In the strictest sense, a "processive" enzyme
incorporates more than one nucleotide before dissociating and a
non-processive ~or distribu~ive) polymerase dissociates from the
template-primer after every addition of a new nucleotide. The
concepts of "processivity'~ and "high processivity" are discussed
in A. Kornberg and T. Baker, DNA RePlication (2nd Ed., W.H.
Freeman and Co., N~Y., l99~) p. 494.
Enz~me catalyzed RNA synthesis proceeds along similar lines
although template ar.d primer requirements are somewhat different,
and riboNTP's are employed as precursors, all as well-known in
the art. Typical RNA polymerases include bacterial RNA
polymerases, eucaryotic RNA polymerases I, II and III, and RNA
replicases, for example, QB replicase.
Fluorescence-labelling has been accomplished in the prior
art by partially substituting a fluorescence-la~elled NTP analog
so that during synthesis an analog molecule occasionally replaces
a norm~l NTP precur~;or in the sequence. Various fluorescent dNTP
derivativ s are known in thP art, having a fluorophore, such as
fluorescein or rhodamine, covalently linked to the purine or
pyrimidine base by a link~r group. Examples of prior art
, fluorescent dNTP derivatives arP ~hodamine-6-dATP, Rhodamine-6-
dCTP, Fluorescein-7-dUTP and Fluorescein-12-dUTP. The latter is
avail~ble from Boehringer Mannheim Biochemica.
The fluorescent NTPs used in the prior art have been
considered likely to interfere with synthesis due to the steric
interference of the large fluorophore moiety. For synthesis of
` WOg3/19206 CA2i 175~2 ~CT/US93/0~422
DNA longer than 200 bases fluorophore-substituted dNTPs have been
employed as partial substituents for the normal dNTP, i.e., the
syn~hesis reaction mixture contains less than 1.0 mole fraction
of total dNTP as its fluore~icent derivative. For example, a
typical reaction mixture might contain a 1:3 mole ratio of Fl-12-
dCTP:dCTP.
Newer methods of DNA saquence analysis have generated a need
for DNA having at least one of the four dNTPs completely
substituted by a fluorescence-labelled dNTP (dNTP-X). For
example, Jett et al. U.S. patent no. 4,962,037 have developed
techniques for sequentially analyzing the exonuclease produc~s
of a single DNA molecule. Each deoxynucleotide labelled with a
fluorophore is detected in sequence as it is released by
exonuclease action, using a sensitive flow-fluorometric
technique. The efficiency o~ the foregoing technique will be
; improved by providing fully fluorescent labelled DN~ of greater
length than available heretofore.
` ~ .
Methods for synthesizing DNA having at least one dNTP fully
~ubstituted by a fluorescent dNTP (dNTP-X) have been hampered by
~20 the ~teric hindrance as~ociated with in~orporating such
~ derivatives into a DNA molecule. Synthesis of DNA strands of up
;; to~500 bases in length has been reported u~ing dNTP-X having a
linker of less than 8 atoms length between the nucleotide base
moiety and the fluorophore. ~Je t, et al., U.S. Application
Serial No. 07/765,277.)
: :
SUMMARY OF THE INVENTION
:~
The invention provides a method ~or synthesizing nucleic
acids wh rein at least one of the four NTPs is completely
substitutsd by a fluorescent-labelled ribo- or
deoxyribonucleotide, r- or dNTP-X. In one embodiment, labelled
DNA o~ at least 200 bases length can be synthesized. In a
pre~erred embodiment, labelled DNA of at least 500 bases length
can be synthesized. The method includes th~ use of dNTP-X
~ .
,:
W093/192Q6 ~1' 2 1 1 7 5 ~2 PCT/US93/0~422
compounds having the nucleotide ~ase moiety covalently joined to
the fluorophore by a linker chain of 8 to 12 a~oms length. In
the preferred embodiment, such linker chains are those lacking
an ether linkage. In another preferred embodiment, a processive
DNA polymerase is employed. In another preferred embodiment,
single strand DNA binding protein is provided in the reaction
mixture.
Novel compounds suitable for use in synthesizing fully
labelled fluorescent DNA are disclosed herein. These include
Rho-8-dCTP, Rho-lOtJ~-dCTP, Fl-lO(J)-dCTP, Rho-15-dCTP, Fl-15-
dCTP, ~ho-12 dUTP, Fl-12-dUTP, Fl-8-dATP, Rho-8-dATP, Fl-15-dATP,
Res-10-dUTP, HC-6-dUTP, and Rho-12-dUTP (R). In preferred
compounds of the present invPntion, linkage of the f luorophore
does not modify a site on the purine or pyrimidine base that is
normally involved in the hydrogen bonding interactions of base-
pairing. By combining a preferred fluorescent dNTP, single-
strand binding protein and a pre~erred polymerase, the method of
the presen inv ntion has successfully produced fully single-
labelled fluore~cent DNA of greater than 7000 bases length.
The method of the invention is useful to provide fully
: labelled fluorescent RNA or DNA for sequence analysis, for
-~ his~tochemical fluorescent labelling and for micro-analytical
techniqu2s where highly fluorescent RNA or DNA of specified
sequence is desired. A useful kit for carrying out the me ~ od
of ~he invention is also provided.
DETAILED DESCRIPTION OF THE INVENTION
The method of the invention uses conventional enzyme-
catalyzed RNA or DNA synthesis reactions in which the
complementary sequence of a template nucleic acid strand is
synthesized in a reac~ion mixture csntaining an RNA or DNA
pslymerase enzyme, four ribo or deoxy nucleotide triphosphates
(r~ or dNTPs) and optionally an oligonucleotide primer, depending
on the specific requirements of the enzyme chosen to catalyze
synthesis. The template nucleic acid is usually cloned or
~A2i 115~2
W0~3/19206 PCT/US93/02422
purified DNA or RNA whose sequence is to be determined, or for
which a fluorescent complement is desired. The template DNA is
usually in single-stranded, denaturèd, partially single-stranded,
or partially denatured form depending on the template
requirements of the polymerase employed. Primer DNA is typically
an oligonu~leotide whose sequence is complementary to a segment
of the template nucleic acid. 5ynthesis is considered to proceed
by stepwise addition of ribo- or deoxynucleotides to the 3'-end
of the primer, thereby extending the length of the primer, with
concomitant release of a pyrophosphate from the precursor r- or
dN~P. The order of addition of r- or dNTPs is dictated by the
template sequence, such that the newly synthesized ~NA or DNA has
a sequence complementary to the template sequence arcording to
the known base-pairing relationships, A with T (or U) and G with
C, of nucleic acids.
Enzy~es which catalyze DNA synthesis are termed DNA
polymerases. Many DNA poly~erases are known, for example T5
polymerase (Cha~terjee, D., U.S. ~atent 5,047,342), T7
polymerase, E. ~oli polymerase Klenow fragment, Taq polymerase,
Vent7M (New England Biolabs) polymerase and PRDl
polymerase(SaYilah~i, H. et al. (l99l) J. Biol. Chem. 266:18737-
l8744). Reverse ~ranscrip~ases, which catalyze ~NA synthesis
using a RNA te~plate, are also suitable ~or the present
in~entiQn. A wide selection of such enzymes is commerc~ally
available, including AMV-RT and M-MLV-RT (GIBCO BRL), and HIV-RT.
Highl~ proc2ssive enzymes are preferred herein. A "highly
processive" enzyme i5 herein defined as one that incorporates 50
or more nucleotides before dissociating from the template-primer
complex under a given set of reaction conditions. Highly
,30 proces~ive DNA polymerases include phage T5 polymerase and
derivatives thereof ("T5"), phage T7 polymerase and derivatives
thereof ("T7"), phage Phi-29-type polymerases ("Phi-29"), and E.
coli pol III holozyme. T7 DNA polymerase (also called "T5 gene
5 protein") by itself is a DNA polymerase with low processivity.
In the presence of thioredoxin cofactor, however, T7 becomes
highly processive, incorporating thousands of nucleotides from
WO93/19206 C A 2 i 1 7 5 ~ 2 PCT/~S93J02422
a given primer without dissociation (S. Tabor et al. [1987] J.
Biol. Chem. 262:16212; S. Tabor and C. Richardson, U.S. Patent
4,795,699). As used herein, the Phi-29-type polymerases include
Phi-2~, Cp-l, PRD1, Phi-15, Phi-21, PZE, PZA, Nf, M2Y, BIO3, SF5,
GA-l, Cp-5, Cp-7, PR4, PR5, PR722, AND ~7 ~Blanro et al., U.S.
Patent 5,001,050). E. coli pol III holozyme is a highly
processiYe enzyme (processiYity value graater than 5000), as
described by A. Kornberg and T. Baker in DNA Replication (2nd
Ed., W.H. Freeman and Co., NY, 1992), at pages 494-495.
Commercially available RNA polymerases include SP6 RNA
polymerase, T3 and T7 RNA polymerases.
In the synthesis of unlabeled ~NA, the four precursor dNTPs
are d~TP, d~TP, dCTP, and dTTP or, alternatively, dUTP, usually
provided in approximately equimolar amounts with one another.
Either dTTP or dUTP can be used in a DNA polymerase-catalyzed
synthesis of DNA, and for the purposes of the present invention
dTTP and dUTP ~an, as a practical matter, be used
interchangeably, as can their respectiv~ fluorescen~e-labelled
derivatives. In prior art fluorescence-labelling reactions, one
of the dNTPs is partially s~bstituted by a fluorescence-labelled
derivative. For example, the mole ratio of dTTP or dUTP to Fl-
dUTP would typically be about 2:1. As a result, the reaction
produ~t would contain Fl-dU in some sequence loci where dT or dU
would be incorporated. However, the ratio of Fl-dU to dT ~r ~U
Z5 in the product might be lower than that of the reaction mixture
~ecause re ction kine~ics would favor incorporation of dT or dU
over the derivative.
Fluorescence-labelled r- or dNTP derivatives are herein
, abbreviated r- or dNTP-X. The r- or dNTP-X compounds of the
present in~ention have two basic components, a fluorophore and
a linker. The fluorophore can be any highly fluorescent
compound, including, without limitation, fluorescein, rhodamine,
resorufin, and hydroxycoumarin. A -~ariety of fluorophores having
diff erent excitation and emissisn maxima are desirable, in
applications where more than one r- or dNTP is ~abelled. The
` w093~lg206 CA2j i~582 P~T/US93/0~422
linker is typically a cha~n of qreater than 7 and preferably 8
to 12 atoms covalently joining the purine or pyrimidine base of
the r- or dNTP to the fluorophore. When the fluorophore is
resorufin or hydroxycoumarin, the linker can be shorter,
preferably 5 atoms length. The linXer can be an aliphatic chain
of C-C bonds, optionally combined with alkene groups, amide
bonds, ether groups and the like. ~owever, for synthesis of DNA
of greater than 500 bases lPngth, r- or dNTP-X compounds lacking
ether groups in the linker are preferred. It is preferred that
~he linker joins the purine or pyrimidine moiety at a site or
atom not involved in the hydrogen bond formation of nucleotide
base pairing. For adenine, these sites are the 6-amino group and
the l-N of the purine ring; for guanine, the 6-OH , the 1-N and
the 2-amino group; for cytosine, the 4-amino group, the 3-N and
the 2-OH; and for thymine, the 4-oxo group and the 3-N.
The structures of dNTP-X compounds useful in the practice
of the synthesis method are shown in Tables 1-4. The
nomencla~ure used herein identifies the fluorophore, the linker
length (except in the case of ~C 6-dUTP and Res-10-dUTP) and the
nucleotide base. HC-6-dUTP and Res 10-dUTP, as used herein,
refer to the commercial names for hydroxy~coumaxin-6-dUTP and
resorufin-10-dUTP, respectively. Abbreviations for fluorescein,
rhodamine, resorufin, and hydroxy-coumarin are Fl, Rho, Res, and
HC, respec$ively. For example, Rho-12-dUTP is dUTP joine~ to
rhod~mine by a linker chain of 12 a~oms. Use of a ~effamine
precursor for syntheæis is indic:ated by including " (J) " in the
abbre~iated name and also indicates the presence of ether groups
in the linker. Use of an "(R)" immediately after the abbreviated
name indicates a rigid linXer as compared to the non-rigid
,30 analog; e.g., Rho-12-dUTP(R) versus Rho-12-dUTP~ Specific
~luorescence-labelled nucl~otides are designat~d herein as dATP-
X, dGTP-X, dCTP-X and dUTP-X.
CA2i 1 7502
W~93/19~06 PCI~/l S93/02422
TABLE 1
O O
H N 1 ~ \i'~N H Jl` R
o N
X
Rho-5-d~rP: R = TETRAMETHYL--RHODAMINE
Rho-12-dUTP: R = (CH2) ~;--NH--CO--TETRAMETHYL-RHODAMINE
:: Rho 12--dUTP ~P~3: R ~ ~CH2~ CH2~ CO-TEq~ETHY~--:RHODAMINE
HC--6-dUTP: R -- ~
o
Re~-10-dUTP: R = {~ ~
F1~5 dUTP: R 3 FLUORESCEIN
: ~ .
:
Fl--I2-dUTP: R -- (CH2)s--NH--CO--FLIJO~ESCEIN
X = DEOXYRIBOSE--5 '--TRIPHOSPHATE
:~
W093/19206 CA 2 j 1 7 5 8 2 PCT/US~3/0242
TABLE 2
NH /~NH~R
: 1 11
~: N~ O
0~1
: X
::
: : :
:
: Rho-8-dCTP: R = T~TRAMETHYL~RHOD~MINE
~18-dCTP: ~ = FLUORESCEIN
~, .
Rho-15-dCTP: R = -( ~ )5NH-CO-TETRAMETHYL-~HODAMINE
Fl 150dCTP: R = -(CH2)5NH-CO-FLUORESCEIN
X c DEOXYRIBOSE-5' TRIPHOSPHATE
~'A~J; `I / '32
WO 93/1~206 PCr/US93J~242
TABLE 3
NH --o~NH~R
N~
X
~::
.f~
Rho--10 (J) -dCTP: R = TETRA~![ETHYL RHODAMINE
Fl--10 (J)--dCTP: R = ELUORESCEIN
X = DEOXYRIBOSE--5 '--TRIPHOSPHATE
l il 2 i i l ~ ~ ~
YVO 93~19206 PCI /US93/02422
TABLE 4
NH ( CH2 ) 6NH-CO-R
N~N
~N N
~.
~ ~ ~ ,f"
:
Rho-8--d~TP: R = TETR~MET~HYL--RHODA~!lINE
Fl--8 -dATP: P~ -- F~UORESCEIN
Fl--15 ~TP: Pc = (CH2)5--NH--CO-FLUORESCEIN
X = DEOXYRIBOSE--5 ' -TRIPHOSPHATE
WO93~19206 C~ 2 i , /5 8 2 PCr~US93/02422
Because of the prior art practice of par~ially substituting
a dNTP-X for the corresponding dNTP in a DNA synthesis reaction,
specific distinguishing terminology is used herein. Complete
substitution for one of the r- or dNTPs means that on~ of the r-
or dNTPs is replaced entirely by an r- or dNTP-X. For example,
complete substitution of dCTP by dCTP-X means that the reaction
mixture contains dATP, dGTP, dUTP and dCTP X, but no dCTP
(within the practical limitations imposed by the purity of the
dCTP-X preparation~. The DNA synthesized in such a reaction
I0 mixture is termed herein fully single nucleotide lab~lled DNA.
Every cytosine (C) residue in such DNA would be replaced by C-X,
a fluorescence-labelled cytosine, except for that portion
represented by ~he original primer. Fully double nucleotide
labelled DNA is then DNA in which two of the four dNTPs are
completely substituted for by their corresponding dNTP-X
derivatives. Similarly for fully triple and quadruple
nucleotide-labelled DNA.
Addition of single-strand binding protein to the reaction
mixture has been found to enhance yield and to increase the
maximum length of fully labelled product. A total yield increase
of over 200% was ob~ained in some instances.
Complete substitu~ion of a dNTP-X results in inhibition of
the DNA synthesis reaction. The extent of inhibition varies with
the polymerase used and the dNTP-X. Inhibition from 40% to 9~
relative to csntrol reactions using the unmodified ~NTPs was
observed. In general reactions inhibited more than 95~ failed
to yield fully labelled DNA of greater than 500 bases length.
The extensive inhibition nbserved with complate substitution may
, have been a significant factor in directing the art away from
complete substitution heretofore.
A kit combining the main components for carrying out the
invention is also provided. The kit includes a DNA polymerase
enzyme and an assortment of dNTPs and dNTP-X substrates. In one
embodiment a kit designed for complete substitution of one dNTP-X
` WO~3/192~6 C~2 i 1 7582 PCT/USg3/0~422
will contain 3 dNTPs and one dNTP-X, either separately packaged
or premixed. In another embodiment, providing greater
flexibility, a complete set of dNTPs and dNTP-X derivatives is
provided, to facilitate any desired combination of complete
single, double or multiple fluorescence labelling. Such kits
optionally provide a standard template DNA and standard primer
for control and calibration, howe~er, the end u~er will provide
the desired template and primer for the specific desired purpose.
The novel compounds of ~he invention and the method of DNA
synthesis using said compounds, or known compounds having the
described chara~teristics, is described in the following
examples. All me~hods and procedures not otherwise described
herein were carried out using published methods, as referenced,
or by techniques well-known to those of ordinary skill in the
art. Standard abbrevia~ions are used, unless otherwise noted.
The principles and teachings disclosed herein with respect to DNA
synthesis apply also to RNA synthesis, with appr~priate
modifications of reaction conditions, adapted to RNA synthesis
and RNA synthesis-catalyzing enzymes, as are well-known in the
art.
EX~MPLES
Exam~le 1: SYnthe~is of Modified Nucleoside TriPhos~hates
1. Amino-nucleoside tr ~hosphates. ~
N4-Aminohexyl dCTP (precursor of Rho-8-dCTP, see Table 5)
and N4-Jeffamine-dCTP (precursor of Rho-lO(J)-dCTP and Fl lO(J)-
dCTP) were prepared by the transamination method of Draper (1984
Nucl. Acids Res. 12:989-1003.
,
N6-Aminohexyl dATP (precursor of Fl-8-dATP and Rho-8-dATP)
was prepared by the method of Gebeyehu e~ al. (1987) Nucl~ Acids
Res. 15:4513-4534.
13
CA21 1 75~2
WO 93/19206 PCI`/US93/0242?
Allylamine dUTP (precursor of Fl-12-dUTP, Rho-12-dUTP and
Rho-4~dUTP) was prepared according to L~nger et al. ~1981~ Proc.
Natl. Acad~ Sci USA 78:6633-6637.
2. Labellinq of amino-nucleoside triPhosphates
S with fluores~ent dves.
The amino-nucleoside triphosphates (10-20 ~mol) were
dissolved in sodium bicarbonate ~0.4 M, 500 ~1) or sodium borate
solution (0.1 ~ and treated with a 3 to 5-fold molar excess of
the N-hydroxysuccinimide ester of thP dye in anhydrous dimethyl
formamide ~500 ~1). The mixture was rea~ted for 3 - 18 hr. at
xoom temperatur~. The reaction was monitored by thin layer
chromatography (silica gel; butansl: ace~one: acetic acid: 5%
ammonium hydroxide: water/70:50:30:30:20~ and~or ~y HPLC. The
crude mixtures wer~ diluted in water (200 - 300 ml), loaded on
1~ a 10 - 15 cm long, by 1 cm diameter column of mild anion exchange
resins and eluted, sequentially, with 0.01, 0.2 and 0.5 M
~: triethylammonium bicarbonate until ~he fraction containing he
fluorescent dNTP was collect d. After desalting of the
appropriate column fraction, TLC, HPLC and capillary
electrophoresis analysis was used to as~ess t~e purity and
characteristic elution pattern.~ of the desired product. The
compounds were characterized by their U.V. spectra as the
: overIapping spectra o~ the starting amino modified base an~t~e
: dyes. Yields o~ fluorescent nucleotides were 50 - 60~.
1:
WO93/19206 CA2i i7~r,~2 PCIJUS93/02422
~ ~-: a z 4 a z o o o o a o o
_. _ _ o o _
X X N N C~ g n o A A N n N N
a --o _ _ o- O O O O OA O
.. C O N Z V V O O O N O O N
z o n v a n n IA n o n n ~ v
~ ~ Z: ~ __~ ~ u u~ ~ ~ r~ ~ _
_ 3 o o o o o o z o o z a z
~ _, o V o o s~ o o
E~ _ _ _ _ o o o _ ~ _ .~
~ ~ X N n n n n o o o n o o o
E-' Ul Ul ~ 5'~1 t`J ~ It~ ~ tf) N N ~1 ~)
~Jv) _ _ _ _ --
L-- ~ ~A O IA N = N ¦ IA A ~ = IA C
_ _ _ _ C~0
~,3 ~ ~ f' E~ ~ E~ ~ E~ ~ ~ ~ E~
~ ~ o~ o ~ ~ ~ ~ aJ ~ o ~ a ~ ~
,, ' . __,.. G _ __ h L_ O O ._ _ O N
WO~3/19206 C A 2 ! i 7 5 ~2 PCr/US93/02422
Example 2: In Vitro Synthesis of Fluorescent DNAs.
The fluorescent dNTPs used in these s~udies are shown in
Table l. The number in the abbreviated name of each compound
refers to the length of the linker which joins the fluorphore to
the base moiety of the dNTP.
Fluorescent dNTPs were synthasized by standard methods. The
dNTPs listed as "Orange" or 1'Green" contain modified Rhodamine
or Fluorescein dyes, respectively and arQ proprietary products
of Imagenetics, Inc., Naperville, Ill.
The following DNA polymerases were tested: 1) T5 DNA
pol~merase modified to lack 3' to 5' exonuclease acti~ity
~[T5(Exo-), BRL]. 23 T7 DNA polymerase modified to lack 3' to
5' exonuclease activity tT7(Ex~-); U.S Biochemical~. 3) The
Kl now fragment af E. li DNA polymerase I (BRL). 4) T.
aquaticus DNA polymerase (Cetus). 5) T. litoralis DNA
polymerase (Vent~, New England Biolabs). 6) Phage P~Dl DNA
polymerase.
These DNA polym rases are typical of the following general
types of enzymes: l~ Highly processive E. coli enzyme
(T5(Exo-), T7tExo-), Phi-29-type such ~s P~D-l, and E. coli-pol
III holozyme; 2) E. coli enzyme of low proces iYity (Klenow
fra ~ en~); 3~ Thermostable enzymes ~Taq, VentTM, VentS . XO-)T~.
Prior to performing exp~riments with any given DNA
polymera~e and fluorescent ~NTPs, reac~ions as described below
were run containing increasing amounts of polymerase in the
presence of the 4 normal dNTPs and a constant amount of template
and primer. The amount of reaction product was ~uantitated by
TCA precipita~ion of radioactive molecules. A concentxation of
polymerase that gave a plateau of activity in this assay was used
in assays described below that test the ffects of fluorescent
dNTPs on synthesis. In addition, control experiments which
16
WO93/1~206 CA 2 1 1 7 5 82 PCT/U~93/02422
lacked, respectively, primer, template or single individual dNTPs
~e.g.f dCTP or dTTP) were run to prove that synthesis in the
reactions described below was dependent on the presence of
primer, template and all 4 dNTPs and thus represents copying of
the DNA template by ~he DNA polymerase rather than some
artifactual nonprimer-dependent process.
A typical reaction in which the effect, for example, of
Rhodamine-8-dCTP on synthesis of DNA by T5(~xo-) DNA polymerase
was determined consisted of the following:
In a total volume of lS nicroliters: 27 mM Tris-HCl, pH
7.5, 13 m~ MgCl2, 33 mM NaCl, 7 mM dithio~hreitol, 1 ~g Ml3mpl9
Strand DNA, 5 ng 23 ~p pximer (BRL), lO0 ~ each, dATP, dGTP,
dTTP, 100 to S00 ~M Rho~8-d~TP and 2 ~ci/ 32P-dGTP. The reaction
was incubated at 37C for l hour and 8 ~l were removed. Of this,
: 15 3 ~l were TCA precipitated to test for he effect of the
: fluorescent dNTP on acti~ity and the remaining 5 ~l were run on
an alkaline agarose gel according to the procedure of Maniatis
et al. (l982) "~olecular Cloning, A Laboratory Manual", first
edition. Radioactive size markers (1 KB ladder, BRL) were also
run on the gel to allow comparison of the apparent mol~cular
weight of the reaction products with the standards following
a~toradiography of ~he gel. In addition, fluor~scent DNA
molecules in the gel could be visualized directly by placing~t~e
gel on a U.V. light transilluminator. The fluorescence pattern
parallelled the radioactivity pattern. The remainder of the
reaction was incubated overni~ht at 37C and aliquots assayed by
TCA precipitation and alkaline agarose gel electrophoresis, as
above.
After a number of preliminary experiments using this
protocol were performed, a standardized battery of tests was
per~ormed using this basic assay system. These experiments
systematically examined the effects of the fluorescent dNTPs of
Table 1 on synthesis of DNAs by the polymerases listed abo~e.
The precise buffer components and concentration of normal dNTPs
C h ~ 2
W093/1~206 PCT/US93/02422
were varied to be at the optimum ~or ea~h enzyme, as indicated
by preliminary experiments or by data from the manufacturer~
Reactions with ~he thermal stable polymerases, Taq, VentTM and
Vent(Exo-)T~, were run at 72C, as recommended by the
manufacturer.
The reaction conditions for each of these experiments was
as follows. All reactions were per~ormed in a final volume of
15 microliters containing 1 ~g M13mpl9 ~ s~rand DNA, 5 ng 23 bp
primer, 2 ~Ci 32p dGTP, and, respectively in two different
reactions, 100 or 500 ~M of ~he ~luorescent dNTP, and the
specific buffers, concentrations of normal dNTPs and enzyme units
listed below. The same results were obtained at either
conce~tration of the fluorescent dNTP. Rpactions wPre performed
for 1 hour and overnight at the given tempexature ~see below) and
assay~d by TCA precipitation and alkaline agarose gel
electrophoresis, as described above. Other aspects of the
reaction conditions were:
1. For ~5 (Exo-) and T7 (Exo-) DN~ polymerases: Normal
dNTPs (100 or 200 ~M; results id ntical), Reaction buffer ~as
ZO above), Reaction temperature (37C), Enzyme (1 unit).
2. For Taq DN~ polymerase: Normal dNTPs (200 ~M~, Reaction
buffer (10 mM Tris-HCl, pH 8.3, 2 ~M M~C12) Reaction tempera~ure
~72C), ~nzyme (2 unîts).
3. For VentTH and Vent (Exo-)T~ polymerases: Normal dNTPs
(200 ~M), Reaction buffer (10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-
HC1, pH ~.8, 8 mM Mg2(S04)2, 0.1% Triton X-100, 100 ~g/ml bovine
serum albumin), ~eaction tPmperature (72C) , Enzyme (1 unit~.
4. For Klenow fragment of E. coli DNA polymerase: Normal
dNTPs (100 ~M), Reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM
MgCl2, 6.7 mM dithiothreitol), Reaction temperature (37C~,
:~nzyme ~1 unit).
18
W093/19206 C ~ 2 1 i ~ ~ ~ 2 PCT/US93/02422
5. For PRDl polymerase normal dNTP's (200~M), Reaction
buffer t5mM Tris-HCl, pH 7.S, 7.5 mM MgCl2, O.5 mM
dithiothreitol, O.S mg/m~ BSA), Reaction temperature (37C),
Enzyme (1 unit).
\
The most import nt parameter ~or utility of a fluorescent
dNTP is the size rang~ of products that it gives with different
polymerases. These results are summarized in Table 6 for
overnight enzymatic reactions.
~Overnight reaction~ were usually performed for convenience.
DNA molecules grPater than 500 bases are synthesized, for
example, by T5(Exo-) polymerase after 1 hour of reaction. The
average size of the reaction products increases for at least 6
hours at 37C.
Data from TCA precipitations (not shown) indicated tha~ the
~15 various fluorescent dNTPs inhibited synthesi, of DNA from ca. 40
;: to 99% relative to control reactions containing the 4 unmodified
: dNTPs. In general, ~hose reactions that were inhibited not more
than ca. 95% produced some fluorescent DNA molecules gr~ater than
500 bases in siæ~. There was, in general, a strong correlation
betwe~n degree of inhibition and size of the fluorescent reaction
products.
,f~
: To determine whether two differ~nt fluorescent dNTPs could
~e incorporated in the same reaction, T5(Exo-) and ~7(Exo-)
polymerases were tested with combinations of fluorescent dNTPs,
:~:25 as shown in Table 6. The overnight reactions were carried out
exactly as des~ribed above except that both of the two
fluorescent dNTPs were present at lOO ~M and the two remaining
normal dNTPs were present at 200 ~M concentrations. As shown .n
Table 6, only certain combinations of the dNTPs gave products
greater than 500 bases in size.
19
WO93/19206 C ~ 2 1 1 7 5 8 2 PCT/US93/02422
_
Table 6: Size of fluorescent DNAs containiny two different
fluorescent labels as assayed by alkaline agarose gel
electrophoresis.
DNA Polymerase:
T5(Exo-) T7(Exo-)
Fl 12-dUTP _
Rho-8-dCTP 500 - 1~00* 300 500
_
Green-dUTP
Rho-B-dCTP 5~0 - 1000 300 - 600
.
.
Fl-12-dUTP
~ho-10(J)-dCTP 200 - 300 200 - 300
Fl-12-dUTP
Green-dCTP 200 - 500 200 - 300
. _ _
Green-dU~P
Green-d~TP 200 - 300 200 - 300
_ ~
* Sizes are given in bases
- -- f ~
Th~ following conclusions were drawn from these studies:
1) Hi~hly ~rocessiYe DNA polymerases such as T5tExo-), T7(Exo-)
and P~D~ are particularly advantageous for synthesis of
fluorescent DN~s. T5(~xo-) DNA polymerase is the best enzyme
thus far examined for this purpos 2~ Fluorescent dNTPs
containing linkers longer than 4 atoms (and preferably ca. 8-12
a~oms) attached to non-hydrogen bonding positions are ~he best
dNTP-Xs. 3) The chemical structure of some linkers (e.g., that
of Rho-lO(J) dCTP and Fl-lO(J()-dCTP) inhibits synthesis even
though the linker is long. 4~ The combination of Rho-8-dCTP,
F1-12-dUTP and T5(Exo-) DNA polymerase allows synthesis of fully
s
W093/19206 C~ 2 i i 7582 P~T/US93/0~422
double nucleotide-labelled fluorescent DNAs greater than 500
~ases in size.
Example 3: Stimulation _ of Fluorescent DNA _Synthesis by
Sinqle Strand DNA Bindinq Protein.
The following reaction was performed. Fifteen microliter
reactions, basically as describ~d above for T5~Exo-) DNA
polymerase, were per~ormed which contained 1 unit of T7~Exo-) DNA
polymerase, 0.25 or 1.O ~g of Ml3mpl9 ~ strand template,
respectiYely, 100 ~M Rho-8-dCTP, lO0 yM dATP, dTTP and dGTP and
6 ~g of Single Strand ~inding Protein ~purchased from Pharmacia).
The Single Strand Binding Protein enhanced synthesis of
radioactive, fluoresc~n~ DNA by 205% and 168%, respectively, with
the two amounts of template in a one hour reaction relative to
a rea~tion that contained Rho-8-dCTP but no Single Strand binding
Protein. Most importantly, in overnight reac~ions, reactions
containing Single Strand Binding Protein contained fluorescent
D~ of si~es 500 ~p to 5 ~b as compared to 500 bp to 2 kb in
reactions lacking the protein. This proves that Single Strand
Binding Protein can significantly enhznce the synthesis of
fluorescent DNA.
Example 4: Synthesis of Fluorescent Probes for In Si~u
H~bridization. ~
That probes suitable for in situ hyb~idization can be
produced using direct incorporation of fluorescent nucleotides
was shown by the following experiment. A synthe~is reartion,
similar to that described above, was performed using Rho-8~dCTP
as the fluorescent dNTP, human Cot-l DNA as template (Human Cot-l
DNA is total human genomic DNA greatly enriched for highly
repetitive sequences and was purchased from BRL), random
hexanucleotide primers (BRL) and Klenow DNA polymerase. The
Human Cot-l DNA will potentially hybridize to human ~NA present
in rodent-human hy~rid cell lines, thus allowing the
identification ~f the particular human chromosome contained in
the rodent cell. To ~est this, the fluorescent DNA copy of the
WO~3/19206 C ~ 2 i i 7 5 8 2 PCT~US93/02422
human Cot-l DNA was hybridized to metaphase spreads prepared from
the WA-17 cell line, which contains human chromosome 21 in a
mouse chromosome background, by standard procedures. The single
copy of Human Chromosome 21 pr~sent in the cell line was readily
detected by fluorescence microscopy and mouse chromosomes were
not labelled by the probe. Other experiments indicated that the
fluorescent Cot-l DNA probe hybridizes to all of the human
chromosome present in metaphase spreads prepared from human
peripheral lymphocytes.
Example 5: Effect of dATP Derivatives on Polymerases
1. Klenow DNA PolYmerase
The effect of dATP derivatives on direct incorporation by
Klenow DNA polymerase was shown by the following experiment.
Synthesis reactions, similar to that described in Example 2
above, were per~ormed using normal dATP and the dATP derivatives
shown in Table 7. Note that "HC-6-dUPT" and l'Res-10-dUTP" refer
to the commercial n~mes for ~ydroxy-coumarin-6-dUTP and
Resorufin-10-dU~P, respectively, and thus the numbers in the
a~breviated names do not refer to the length of the linker for
these compounds.
Fifteen microliter reactions were performe~ which contained
1 unit of Rlenow E. coli DNA polymerase, 1 ~ M13mpl9 ~ Strand
DNA, 5 ng 23 pb primer (BRL), 100 ~M each dCTP, d&TP and dTTP,
reaction buf~er, 2 ~Ci 32P-dGTP, and, respectively in two
different reaction~, 25 or 100 ~M of the normal dATP or dATP
derivative. Reactions were performed for 1 hour at the 37 C and
assayed by TCA precipitation and alkaline agarose gel
electrophoresis, as described above. The re~ults are summarized
in Table 8. As shown by the percent activities, both bio-7-dATP
and AH-dATP are better substrates than the fluorescent d~TPs, and
both produce larger reaction products. Reactions containing bio-
7-dATP produced DNA of sizes 1000-1600 bases and AH-dATP (linker
snly? produced DNA of sizes 500-2000 bases; reactions containing
fluorescent dATPs produced fluorescent DNA of sizes less than 500
WO93/19206 C A 2 i 1 7 5 8 2 PCT/US93~02422
bases. This proves that incorporation of biotin dATP or AH-ATP
is not predictive of the incorporation of the cognate fluorescent
dATPs, nor does ~ynthesis of biotinylated DNA predict the
synthesis of large fluorescent DNAs.
23
W~ 93/19206 (~ i~ 2 1 i i 5 ~ 2 P~r/US93/02422
T~B~B 7
o IIN INH
N H ( C H2 ) ,~-Ni~-C- ~ ~H;~
5 i 3io-7~dATP
<~N
'I NJ
d R
N H - ( C H 2 ~
<f ~M AH~ P
~RTP
NH-( c H2~,-N~
N~J uoJ~ Ft-7
~lu~r~ ein
H h - ~ C H ;~ 3 6 ~ N H~ ao-7
<N~NJ ~ )2
,
T~tra~et~yl:r~od~ine
SUBSTlrlJTF SHEET
W093/19~06 iJ A2 i ~; 5' 2 P~/U~93/û2422
_= =
__ o
o ~ C~
,1 l o o
N O O O O V V
_ U~ G ,_1 ~1 ~1
~:
~r ,1 o
~ '~ O ~ ~ F`~ ~1
: _ . _
1~: ~D O ~1 .~ 1~
ia O H O U~ CD ~ r~
~ O l _ _ _ __
_ In Oo O O
,_ 0 ~ O ~ ~ O
t) Q~ ) O O O ~
~ ~ N O O O O V
O o ~ o o ~n
~; O In ~ .1 ~
~o ~ ~ -- - - -
h ~ o o ~i r~ o ~
H a O o ~l ~ h
O ~ ~ ~ ~e~
_ - _ ~ ~ 0
~4 ~ E~ E~ E~ ~OQ
.~ ~ E~ ~ ~ ~
a: ~ ~ r~ ~ r- r
_ iLI i~ N
C.) o\
~i
WO93/1~206 PCT/~S~3/02422
l A 2 i 1 7 j ~ 2
1. TSrExo ~L___A Polymerase
The ~ffect of dATP derivatives on direct incorporation by
T5 (Exo-) DNA polymerase was shown by the following ~xperiment.
The 1 hour reactions were carried out exactly as des~ribed above
except that T5(Exo-) DNA polymerase was used. The results are
summarized in Table 9O
26
WO ~3/19~)6 ~ PCr/US93/02422
~1 2 i 1 ~82
_ _ __ .
o ~ l l l , o
N O O O O t V
u ~1
.~ ~ ~1
.~ ~ . _
E~
~: ~ ~ ~r o I~ ~
~D
~ _ O ~ ~1 ~ C~ U~
2 v H ~ t~ ir) t~
c¢, O :- _ .
:: ~ . ~1 O O O
~n o o o
O _~ .5J O ~D
O O ~ t l l O O
1::~1) ~ O O O Itl It~
Z V U~ ~ O O O V V E~
O ~
~ ~ . ~ t~ tX~ ~1 -
::~ ~ o .1 ~a o o
: ~ h ~ o m ~ ~1 o
~ ~ d~ ~ S;
~ . . _ _ _ ~ ~
_ S~ O ~D U) O r) a~ 0
H t` N 9~ ~1 ) Q ~U
u) ~: o~ ~ ~1 ~r ~
~ ~ _ ~ ~ ~ C ~
.~ ~ E~ ~ l ~ ~ ~
a~ ~ ~ I~ ~ ~ r ~ r~ ?
E~ O l l O I
E~ ~ ¢ .~ :r: _~ C
fC ~ ~3 ~ ~ ~;4 ~i N ~ ¢
E~ _ _ u~ ~ o~O
27
WO93/19~06 PCT/U~3J02422
~,
CA21 ~i 75~2
The following conclusions were drawn from these studies:
1) T5~Exo-) DN~ polymerase is a better enzyme for incorporating
biotin dATP and AH-ATP than is the Xlenow polymerase, as shown
by the percent activities. 2) Rho-7-dATP at a high
1 5 concentration is a much better substrate ror T5(Exo-) polymerase
~24.6% activity) than i~ is for Klenow polymerase tl.9%
activity). 3) Both bio-7-dATP and AH-dATP produce larger
reaction products than fluorescent dATPs, which produced
fluorescent DNA of sizes less than 500 bases. 4) Incorporation
of biotin d~TP or AH-NTP by a particular polymerase is not
predictive of the incorporation of the cognate fluorescent dNTPs
by that same polymerase. 5) Incorporation of biotin dNTP or AH-
NTP does not predict the differ~nces in incorporation of
different fluoresc~nt dNTPs by different polymerases (see also
lS Table 5 above). 6) Synthesis of biotinylated DNA does not
predict the synthesis of large fluorescent DNAs. 7) A given
fluorescent dNTP is both an inhibitor and a substrate for DNA
polymerases, and different polymerases thus have different Km and
Ki values for a given dNTP. Differences in Km andtor Ki ~alues
Z0 is shown, for exam~le, by comparing the percent activities of
Rho-7-dATP and Fl-7-dATP. Rho-7-dATP is a much better substrate
for the T5(Exo-) polymerase as the concentration is raised from
25 to lO0 ~M; the activity of Fl-7-dATP, on the other hand,
decreases with increasing concentration. 8) The fact ~at
di~ferent polymerases have different Km and Ki values for
fluorescent dN~Ps makes it impossible to predict a priori the
activity of a given ~luorescent dNTP with a given polymerase.
The behavior of fluorescent dNTPs with polymerases cannot be
: predic ed ~rom the behavior of biotinylated nuzleotides, for
example, since biotinylated nucleotides themselves have dif~erent
effects on Km and Xi ~alues.
:
28
WO93/19206 ~ A 2 i 1 7 5 ~ 2 PCT/US93/02422
ExamPle 6: Synthesis of Fluorescent DNAs
The effect of several modified dNTPs on direct incorporation
by various polymerases, including reverse transcriptases and
highly processive enzymes, was shown by the following experiment.
The fluorescent dNTPs used in these studies are shown in Tables
~ and 2. Res-10-dUTP (Resorufin-10-dUTP) and HC-6-d~TP ~Hydroxy-
coumarin-6-dUTP) were purchased from Boehringer Mannheim
: Biochemica. Fl~ dUTP, Rho-12-dUT~, Rho-12-dUTP (R) and ~ho-8-
dCTP were synthesized as described in Example 1. Rho-12-dUTP and
Rho-12-dUTP (R) differ in the structure of the linker, the latter
compound having a more rigid linker than the former. The
reaction conditions were identical to those of Example 2 except
that the reactions were performed overnight with one of the
following polymerases: T5(Exo-), purchased from BRL; T7(Exo-),
purcha ed from U.S. Biochemical; AMV-RT and M-MLV-RT, purchased
: from BRL; and HIV-RT, obtained from Dr. Steven Hughes of the
:~ National Cancer Institute. The three reverse transcriptases
: (AMV-RT, ~-MLV-RT and HIV-RT) copied DNA templates and thus were
us~d as DNA polymerases. Reactions were assayed by TCA
pre~ipitatio~ and alkaline agarose gel electrophoresis, as
described above. The results are suamarized in Table lO.
::
WO93/19206 CA2i i75~2 PCr~US93/02422
_ _
~i O O Q O O O
- ! ~ o o o Y v
I ~ ~r In
I ~ O ~1 ~ O ~D O
U~ I H ~ l I ~ l ~
O ¦ :C V - O O V O V
~ r_ _
~ D~ ~ O O O O O O
: ~ ~ I ~ N O O O O V
H ~ U)U~ IS~ ~
Q~ I_ _ _ _ . ._
tO 1~ I _~ O
V X O t` 00 00
æ tn I ~ OD l ~ ~ z : :z;
e~ ~ I o o o o
I r~ o o o o ,.
E~, ~ ~ ~ ~
___ . I
o o
~ ~ ~ o o o o
V) ~ l o ~ o o
ff'ff o o I~ o ~
Y ~ X f,~ J'~ f~ A f~f Cl t~J~ ff~
o .~qf fff~f l o o l z; z ~ ff
f ~ 1 ~ JSI' f. ,f f_f O O ffa
'~ W f~` fm mf ~f fX' .~Q f'
~ ff',fff~f C iJ'~f
~ I l~ t~
ff~ ~ ~ ~ f-ff ~f
O O.f l f~ l f~ ff~, S~f ~?
~ ~ ff. ~ O ~5 f f 'N r~; CO 'Il~ C
~;f '~5 ~ 'ID l l ~ l fU~f f~f
f ff U~ V S f ff ,~ ff--I C' lf`f f_
ff¢f _ x _ ~ ~ ~ ff~ ~ .fff a
W093~l9206 PCT/US93/024~
The following conclusions were drawn from these studies:
1) HC-6-dUTP is a par~icularly good-substrate with T5~Exo-) and
T7(Exo ) polymerases and promotes the synthesis of large
fluorescent DNAs (some greater than 7 kb in size). 2) The rigi~
linker of Rho-12-dUTP (~) is particularly advantageous for
T5(Exo-) DNA polymerase relative to Rho-12-dUTP. 3) The reverse
transcriptases synthesize fluorescent DNAs greater than 200 bases
in size using certain dNTPs, particularly HC-6-dUTP and Rho-12-
dUTP. Like other polymerases, the reverse transcriptases also
vary in their abilities to incorporate certain fluorescent dNTPs
~see above~.
