 |
Description  |
|
|
The present invention relates to methods of making an oligonucleotide. The
present invention also concerns methods of making oligonucleotide probes.
It has been shown by Studencki and Wallace (DNA 3, 7 (1984)) that an
oligonucleotide can be labeled with radioactive .sup.32 P using an enzyme
and a combination of primer and template. The method is efficient and can
produce highly labeled probe. The method has a serious disadvantage
because the template and the products have identical electrophoretic
mobility unless one is phosphorylated at the 5' end, or the template or
the primer is tritylated at one end. This requires that one of the strands
be labeled with .sup.32 P using terminal labeling enzyme, or tritylated
and 100% of these molecules should carry the modification.
The modification by phosphorylation is not efficient enough to produce 100%
5' end phosphorylated nucleic acids. Moreover, depending on the sequence,
the mobility difference between phosphorylated template extended primer
and the other strand may not be enough to separate them. 5'-end tritylated
residues have the disadvantage of liability in aqueous solutins. The
present invention is a method of separation of primer extended
oligonucleotide probes from the templates by modification of one of the
participating oligonucleotides. The modification is such that it is
stable, hydrophobic/hydrophilic and changes the electrophoretic mobility
and elution property on a reverse phase column and if desired reusable. It
is desirable that the template be modified to carry the separability
property. The synthesized labeled moleculesshould remain unmodified other
than the modifications introduced by an enzyme reaction used in the
synthesis.
As for example, if the template is modified at the 3' end with hexylamino
ATP, the mobility of the template during gel electrophoresis will be
slower than the synthesized labeled product. Similar modifications will
also give rise to separability during chromatography under high pressure
on a reverse phase system. The invention can be further described as
follows:
##STR1##
Instead of modifying the template, if a modified nucleoside triphosphate is
used, a labeled strand can also be separated.
A different kind of extension reaction can be used to label or synthesize a
specific nucleic acid sequence. In Method 1 the template contains the
complete sequence of the nucleic acid to be copied (described in the
diagram) on the template. A template primer pair can be chosen such that
one can be extended to complete a desired sequence without extending the
other of the pair. As for example, the following four pairs will produce
nonadecanucleotide without the use of one of the same length as the
template:
##STR2##
Only the upper strand will be extended if dATP and dGTP are used as NTP
substrates for DNA polymerase or reverse transcriptase reaction. This will
produce 19A'/S' sequence and unextended lower strand.
Similar complementary strands can be synthesized or labeled by using
pyrimidine nucleoside triphosphates instead of dATP and dGTP to produce
complementary sequences. The pairs to be used are
##STR3##
Labeling by using these kinds of pairs is limited to proper sequence
selection. Once they are selected using limited required type and amount
of nucleoside triphosphates one should be able to prepare larger labeled
sequence than the starting reaction partners and hence easy separation
from the reactants. Under certain conditions this separation may not be
necessary for the hybridization assay.
Using nucleoside triphosphates which are to be added to the desired strand,
it is possible to separate the starting materials (unreacted) by gel
electrophoresis or by column chromatography.
In order to maintain the fidelity of the process it is desirable that the
modification of the template be done at the 3' or 5' end. If a primer is
modified its 5' end is the desirable site of alteration. The modification
is carried out via
##STR4##
linkages using known reactions and modified nucleic acid residues at the
specific site. The modifier can be ionic, nonionic, hydrophobic or
hydrophilic. The modification can be before or after the labeling
reactions. If the modification is carried out after the labeling reagent
should be reactive enough to efficiently modify all the desired residues.
As for example, an oligonucleotide with an amine residue (as in
8-hexylamino A or 5-allylamino U containing oligonucleotide) can be
reacted with
##STR5##
similar reagents to form
##STR6##
an oligonucleotide with hydrophobic
##STR7##
residue. A few other typical reactions are described below.
__________________________________________________________________________
##STR8##
X RY XY
__________________________________________________________________________
NH.sub.2
##STR9##
##STR10##
##STR11##
##STR12##
NH.sub.2
isothiocyanate or isocyanate
##STR13##
Epoxide
##STR14##
SH 2,4 dinitrochlorobenzene
##STR15##
Iodoacetamide SCH.sub.2CONH.sub.2
Dansylhydrazine SCH.sub.2CH.sub.2NHdansyl
SHcontaining reagent SSReagent
COOH Alcohol Ester
OH Epoxide ether linkage
##STR16##
Catalytic esterification (Y)
##STR17##
__________________________________________________________________________
EXAMPLE 1
Synthesis of modified oligonucleotide template and modification with a
hydrophobic residue.
##STR18##
Scheme for the synthesis of 1 to be added to 19A' at the 5' end.
THE SYNTHESIS OF 1 IS OUTLINED IN THE SCHEME
5-Chloromercuri-2'-deoxyuridine (compound 5), prepared according to the
method of Bergstrom and Ruth (D. E. Bergstrom and J. L. Ruth, J.
Carbohydrates, Nucleosides and Nucleotides 4 (5), 257 (1977)) was treated
with 3-trifluoroacetamido-1-propene (compound 7) (M. Pailer and W. J.
Hubsch, Monatshefte fur Chemie 97 (6), 99 (1966)) and K.sub.2 PdCl.sub.4
in methanol to give 5-trifluoroacetamidoallyl-2'-deoxyuridine (compound 8)
in 22% yield after 2 chromatographies and a crystallization from methanol.
Reaction of compound 8 with 4,4-dimethoxytrityl chloride in pyridine
afforded compound 9 in 85% yield after flash chromatography (W. C. Still,
M. Kahn and A. Mitra, J. Org. Chem. 43, 2923 (1978)), which was
subsequently treated with N,N-diisopropylaminomethoxy chlorophosphine (L.
J. McBride and M. H. Caruthers, Tet. Letters 24 (3), 243 (1983)) (compound
10) to give compound 1 as a white solid after precipitation from pentane.
A 19-unit oligonucleotide (I) was prepared using a DNA
H.beta.19A': 3'-GA-GGA-CTC-CTC-TTC-AGA-CG-5' (I)
synthesizer; three separate 1 umole batches of each oligonucleotide were
made and each was attached to a solid support and fully protected. The
dimethoxytrityl protecting group was removed from the 5'-terminus and
compound 1 was attached to the 19-unit chain without the DNA synthesizer,
but using the same reagents and conditions the machine typically employs.
The result of this process is an oligonucleotide with a
5'-aminoallyl-5'-(4,4'-dimethoxytrityl)-2'-deoxyuridine unit at the C-5'
end (II).
##STR19##
When portions of each of the modified oligonucleotides were treated with
3% trichloroacetic acid in methylene chloride, the distinctive red-orange
color of liberated dimethoxytrityl cation was observed, indicating that
the 5-allylamino-2'-deoxyuridine moiety had been attached. The
polynucleotide were lastly de-tritylated with brief exposure to 3%
trichloroacetic acid then purified by polyacrylamide gel electrophoresis.
This demonstrates the utility of compound 1 as a synthon for introducing a
5-aminoallyl-2'-deoxyuridine unit at the C-5' terminus of an
oligonucleotide; incorporation of this unit at any position in the
oligonucleotide should be possible using the same methodology.
The process of attaching an analogue of a Bolton and Hunter (A. E. Bolton
and W. M. Hunter, Biochem. J. 133, 529 (1973)) reagent is described in
Scheme 2. Reaction of the amino-functionalized polynucleotide compound 11
with the N-hydroxysuccinimide ester of 3-(4'-hydroxyphenyl) propionic acid
(compound 12) will yield the probe polynucleotide compound 13.
The polynucleotide H.beta.19A' is 19 unit polynucleotides corresponding to
a portion of human DNA which codes for the polypeptide hemoglobin,
specifically that region of the DNA wherein lies the mutation which
manifests itself in the formation of sickle-cell hemoglobin and the
genetic disorder known as sickle cell anemia.
##STR20##
EXPERIMENTAL
In the following experimental abbreviations are used as indicated:
g=gram
HPLC=high performance liquid chromatography
L=liter
mL=milliliter
M=molar
mM=millimolar
N=normal
eq=equivalents
mol=gram molecular formula (moles)
mmol=gram molecular formula.times.10.sup.-3 (millimoles)
aq=aqueous
hr=hour
Infrared (IR) spectra were obtained with a Perkin-Elmer Model 710B or 237
infrared spectrophotometer as solutions in CHCl.sub.3 unless otherwise
noted; the 1602 cm.sup.-1 band of polystyrene film was used as an external
calibration standard. Signals are reported as cm.sup.-1.
Proton magnetic resonance (.sup.1 H NMR) spectra were obtained at 89.55 MHz
using a Varian T-60 spectrometer; spectra were obtained in CDCl.sub.3
solution unless otherwise noted. Chemical shifts are reported in parts per
million downfield from the internal standard tetramethylsilane, unless
otherwise noted.
Carbon-13 magnetic resonance (.sup.13 C NMR) spectra were obtained at 22.5
MHz using a JEOL FX90Q spectrometer with Fourier transform and with full
proton broad-band noise decoupling; spectra were obtained in CDCl.sub.3
solution unless otherwise noted. Carbon shifts are reported in parts per
million downfield from the internal standard tetramethylsilane, unless
otherwise noted.
Phosphorus-31 magnetic resonance (.sup.31 PNMR) spectra were obtained at
36.21 MHz using a JEOL FX90Q spectrometer; spectra were obtained in
CDCl.sub.3 solution unless otherwise noted. Phosphorus shifts are reported
in parts per million downfield from an external aqueous 15% H.sub.3
PO.sub.4 standard.
Optical rotations were obtained on a Perkin-Elmer Model 141 Polarimeter.
Organic reagents were obtained from Aldrich Chemical Company and were used
without purification, unless otherwise noted. Inorganic reagents were ACS
reagent grade from Fisher Scientific Company or other major vendor.
Reaction solvents were ACS reagent grade. Reagents used in oligonucleotide
synythesis were obtained from Applied Biosystems, Inc. Brine refers to a
saturated aqueous sodium chloride solution.
Thin layer chromatography (TLC) was performed using silica gel 60F-254
plates from E. Merck. Column chromatography was performed using E. Merck
Silica Gel 60 (70-230 mesh). All melting points reported are uncorrected.
5-TRIFLUOROACETAMIDOALLYL-2'-DEOXYURIDINE (COMPOUND 8)
A suspension of 5-chloromercuri-2'-deoxyuridine (compound 5) (D. E.
Bergstrom and J. L. Ruth, J. Carbohydrates, Nucleosides and Nucleotides 4
(5), 257 (1977)) (5.56 g; 12 mmol) in HPLC grade methanol (120 ml) was
maintained under an inert gas atmosphere at ambient temperature and
treated with 3-trifluoroacetamido-1-propene (compound 7) (M. Pailer and W.
J. Hubsch, Monatschefte fur Chemie, 97 (6), 99 (1966)) (7.33 g; 48 mmol; 4
eq) and K.sub.2 PdCl.sub.4 (4.28 g; 1.1 eq). The reaction gradually became
black and was allowed to stir for 22 hr. The mixture was treated with
H.sub.2 S gas for several minutes then filtered through Celite, rinsed
with MeOH and evaporated to dryness under reduced pressure from a
80.degree. C. bath to give a crude semi-solid residue (7.0 g). The residue
was chromatographed on a silica gel column developed with CH.sub.2
Cl.sub.2 /MeOH (5:1). The band which stained a blue color with modified
p-anisaldehyde reagent (Thin Layer Chromatography by Egon Stahl, 2nd ed.
Springer-Verlag, New York 1969, page 857) and had an Rf=0.51 (CH.sub.3
CN/MeOH 3:1) was collected and evaporated to dryness in vacuo to give a
colorless foam. The product was crystallized from a minimum of methanol,
filtered, washed with cold CHCl.sub.3 /MeOH (3:1) and vacuum dried. The
mother liquor was worked for a second crop-total yield 1.01 g (22%). A
recrystallization from MeOH afforded the title compound (8) as
analytically pure tiny white needles with mp=183.degree.-4.degree. C.
after drying in vacuo (<1.0 torr) at 64.degree. C. overnight. IR (KBr)
cm.sup.-1 3420, 3260, 1718, 1683 (br), 1560, 1478, 1283, 1190, 1102, 1061,
980, 788, 763, 737; 'HNMR (DMSO-d.sup.6) (Ref.-DMSO-d.sup.6) .delta. 2.13
(d of d, J=6 Hz, 2H), 3.59 (br s, 2H), 3.70-3.97 (m, 3H), 4.25 (br s, 1H),
5.06 (br m, 1H), 5.20 (br m, 1H), 6.05-6.65 (m 4H), 8.01 (s, 1H), 9.60 (br
s 1H); .sup.13 C NMR (DMSO-d.sup.6) (Ref. DMSO-d.sup.6) ppm 162.05,
155.29, 149.50, 138.05, 124.33, 124.14, 109.96, 87.53, 84.47, 70.23,
61.12, 39.93; [.alpha.].sub.D =+8.01.degree. (c=0.87, MeOH).
Anal. Calcd. for C.sub.14 H.sub.16 N.sub.3 O.sub.6 F.sub.3 : C, 44.33; H,
4.25; N, 11.08. Found: C, 44.19; H, 4.10; N, 10.93.
5-TRIFLUOROACETAMIDOALLYL-5'-O-(4,4'-DIMETHOXYTRITYL)-2'-DEOXYURIDINE
(COMPOUND 9)
A solution of compound 8 (0.60 g; 1.58 mmol) in anhydrous pyridine (8 ml)
was maintained under an inert gas atmosphere and treated at ambient
temperature with 4,4'-dimethoxytrityl chloride (0.67 g; 1.25 eq). After
stirring for 18 hr the reaction was poured into ice water (70 ml) with
vigorous shaking. On standing 1/3 hr at 0.degree. a gummy solid separates
leaving a nearly clear solution which was decanted. The solid was washed
once with H.sub.2 O (5 ml) then taken up in CH.sub.2 Cl.sub.2 (10 ml),
washed once with brine (5 ml) then the CH.sub.2 Cl.sub.2 solution was
dried over K.sub.2 CO.sub.3, filtered and evaporated to dryness in vacuo
to give a brownish foam. The crude product was purified by flash
chromatography (W. C. Still, M. Kahn and A. Mitra, J. Org. Chem. 43, 2923
(1978)) on a column of silica gel (Merck, Grade 60, 230-400 mesh, 60 A) (
75 g) developed with 4.0% MeOH in CHCl.sub.3 solvent (1.0 L). Fractions of
ca. 20 ml each were collected in tubes containing pyridine (10 .mu.l) to
inhibit deprotection of the 5'-hydroxyl. Fractions containing the major
product band (RF=0.29; MeOH/CHCl.sub.3 7:93) were combined, filtered and
evaporated to dryness in vacuo to give compound 9 (0.91 g; 85%) as a
slightly yellowish foam. A fraction from the center of the elution band
was freed of solvent, taken up in EtoAc, treated with Norit 211, filtered
through Celite and evaporated to dryness under high vacuum (<1.0 torr) at
64.degree. C. overnight to afford the analytical sample as a colorless
foam with mp=105.degree.-110.degree. C. (dec.). IR (CHCl.sub.3) cm.sup.-1
3370, 2920, 1715, 1695, 1618, 1515, 1470, 1260, 1182, 1045, 842; 'H NMR
(CDCl.sub.3) .delta. 2.38 (br m, 2H), 3.25-3.75 (m, 5H), 3.75 (s, 6H),
4.10 (br m IH), 4.60 (br s, 1H), 5.39 (d, J=16 Hz, 1H), 6.10-6.55 (m, 2H),
6.70-6.95 (m, 5H), 7.15-7.45 (m, 10H), 7.84 (s, 1H); .sup.13 C NMR
(CDCl.sub.3) (Ref. CDCl.sub.3) ppm 162.31, 158.74, 157.70, 156.01, 149.70,
144.04, 137.88, 135.65, 135.52, 130.12, 128.11, 127.26, 125.05, 113.48,
111.33, 86.94, 86.68, 85.25, 72.18, 63.60, 55.34, 42.66, 41.42.
Anal. Calcd. for C.sub.35 H.sub.34 N.sub.3 O.sub.8 F.sub.3 : C, 61.67; H,
5.03; N, 6.16. Found: C, 61.47; H, 5.19; N, 5.95.
5-TRIFLUOROACETAMIDOAMINOALLYL-5'-O-(4,4'-DIMETHOXYTRITYL)-2'-DEOXYURIDINE-
3'-O-(N,N-DIISOPROPYLAMINOMETHOXY PHOSPHINE (COMPOUND 1)
A solution of compound 9 (0.34 g; 0.5 mmol) in anhydrous CH.sub.2 Cl.sub.2
(1.5 ml) maintained under an Argon atmosphere at ambient temperature was
treated first with anhydrous diisopropylethylamine (0.35 ml; 0.259 g; 2
mmol; 4 eq) then dropwise, over 1 minute, with
N,N-diisopropylaminomethoxychlorophosphine (L. J. McBride and M. H.
Caruthers, Tet. Letters 24 (3), 245 (1983)) (compound 10) (0.19 ml; ca.
0.2 g; 2.2 eq). The resultant colorless solution is stirred for 20 min
then transferred with EtOAc (20 ml) (EtOAc was previously washed with
saturated aq NaHCO.sub.3 then brine) to a separatory funnel, washed four
times with brine (35 ml each), dried over N.sub.2 SO.sub.4, filtered and
evaporated to dryness in vacuo to give a colorless glass (0.51 g). This
crude product was taken up in anhydrous benzene (2 ml) and precipitated
into rapidly stirred anhydrous pentane (60 ml) at -78.degree. C. under an
Argon atmosphere. The resulting suspension was filtered, washed with
-78.degree. C. pentane and vacuum dried at <1 torr over KOH overnight to
obtain the title compound 1 (0.38 g; 93%) as a white amorphous powder. IR
(CHCl.sub.3) cm.sup.-1 2965, 1722, 1698, 1618, 1518, 1470, 1262, 1185,
1045, 988, 842; 'H NMR (CD.sub.2 Cl.sub.2) .delta. 0.95-1.30 (m, 12H),
2.20-2.60 (m, 2H), 3.24 and 3.37 (d of d, J=13 hz, 3H) (P-O-CH.sub.3),
3.20-3.80 (m, 6H), 3.75 (s, 6H), 4.17 (br m, 1H), 4.68 (v br m, 1H), 5,42
(d, J=16 Hz, 1H), 6.15-6.55 (m, 3H), 6.75-6.95 (m, 4H), 7.20-7.50 (m,
10H), 7.79 (s, 1H); .sup.13 C NMR (CD.sub.2 Cl.sub.2) (Ref. CD.sub.2
Cl.sub.2) ppm 162.40, 159.21, 157.78, 149.78, 144.71, 138.34, 136.00,
130.53, 128.71, 128.45, 127.54, 125.66, 125.27, 113.82, 111.48, 87.23,
86.31, 85.60, 55.75, 43.78, 43.20, 42.94, 24.99, 24.60; .sup.31 PNMR
(CD.sub.2 Cl.sub.2) ppm 149.30, 148.87, 14.11 (ca. 12% impurity), 8.18
(ca. 4% impurity).
ATTACHMENT OF COMPOUND 1 TO OLIGONUCLEOTIDES
The 19-unit oligonucleotides were synthesized using an Applied Biosystems
Model 380A DNA Synthesizer on control pore glass solid support.
Immediately prior to attaching compound 1 to the 5' end of the oligomer,
the 5'-O-(4,4'-dimethoxytrityl) protecting group was cleaved on the
machine with 3% CCl.sub.3 CO.sub.2 H in CH.sub.2 Cl.sub.2 for 90 seconds.
The support-bound 5'-deprotected oligomer was washed with CH.sub.3 CN and
dried in an Argon stream. Subsequent steps were performed without the
machine, but using the same chemistry;
1. The support-bound oligomer was removed from the container (column) used
for automated synthesis and transferred to a dry septum-cap vial under an
Argon atmosphere.
2. The bound oligomer was treated with a 20-30 fold excess of 0.5M
1H-Tetrazole in anhydrous CH.sub.3 CN followed immediately with a similar
excess of compound 1 in Ch.sub.3 CN. Incubate 30 min with gentle
agitation.
3. Pipette off reagents and wash bound oligomer with 3 portions of CH.sub.3
CN.
4. Treat with an excess of I.sub.2 -H.sub.2 O-Lutidine-THF (0.1M: 1:10:40)
and agitate for 15 minutes.
5. Pipette off reagent and wash bound oligomer with 4 portions of CH.sub.3
CN.
6. Treat with an excess of Thiophenol-triethylamine-dioxane for 60 minutes.
7. Pipette off reagent and wash the bound oligomer with 4 portions of MeOH.
8. Treat with conc. aq. NH.sub.4 OH for 2 hr at ambient temperature.
(Removes protected oligonucleotide from the support).
9. Add more conc. aq. NH.sub.4 OH and heat at 50.degree. C. overnight.
(Removes all protecting groups except the dimethoxytrityl).
10. Filter off the support and evaporate the filtrate to dryness to get
crude oligonucleotide.
This was repeated for all batches of support-bound oligonucleotide.
Treatment of a portion of each on a silica gel TLC plate with 3% CCl.sub.3
CO.sub.2 H in CH.sub.2 Cl.sub.2 produced the orange-red color of
dimethoxytrityl cation indicating the successful incorporation of compound
1 into the oligonucleotides.
One bath of the modified H.beta.19A' oligonucleotide was detritylated with
3% CCl.sub.3 CO.sub.2 H in CH.sub.2 Cl.sub.2 then purified by
electrophoresis on a 20% polyacrylamide gel under denaturing conditions.
EXAMPLE 2
The reaction of a specific oligonucleotide with sulfosuccinimidyl hydroxy
phenyl propionate.
Two micrograms of 19A' amine product of Example 1 is dissolved in 20
microliter of 10 mM borate buffer pH 8.16. To this 5 microliter of a
freshly prepared solution of sulfosuccinimidyl 3-(4-hydroxyphenyl)
propionate (SHPP) purchased from Pierce is added. Concentration of SHPP is
5 mg per ml in water. The reaction is allowed to proceed at room
temperature for 30 minutes then it is kept in the freezer before HPLC
separation is done.
After the reaction, the extent of the reaction is monitored by running a
20% polcrylamide gel in conventional manner.
EXAMPLE 3
Separation of the SHPP reacted 19A' from the reaction mixture containing
hydrolyzed SHPP and oligonucleotides.
HPLC run has been done on a Brownlee RP300 guard column coupled to a
Synchrome RP-P 4.1.times.10 cm column at ambient temperature. A gradient
of (a) 0.1M triethylammonium acetate pH 7 with (b) 0.1M triethylammonium
acetate pH 7.0, 50% acetonitrile at a flow rate of 1 ml/minute. The uv
detector at 254 mm is used to monitor the elution of oligonucleotide. In
order to find out the location of the product, a blank run has been done
with all of the reactants in the starting mixture without the
oligonucleotide. The new peak appeared after adding the oligonucleotide is
taken to be the peak corresponding to the reaction product. After the
product is separated and collected in a fraction collector, the product is
analyzed by gel electrophoresis and from the next run an analytical
determination of the proper peak is not necessary. The yield and recovery
of the product from the columns is between 80% to 90%.
EXAMPLE 4
Primer extension reaction for labeling an oligonucleotide.
The product of Examples 1 or 3 can be used as template and both are
separable by HPLC (high pressure liquid chromatography) or
gel/electrophoresis. An example is provided with the product of Example 1.
An identical procedure can be followed with the product of Example 3.
Reaction conditions have been discussed by Studencki and Wallace, DNA 3
(1984) 7. Primer extension reaction mixture contained 25 .mu.l total
volume and is prepared by mixing to produce a final concentration of 1.6
.mu.M template
(3' GAGGACTCCTCTTCAGACG U*-5') (U* is 5-allylamino U)
and 5.0 .mu.M primer
(5' CTCCTGAGGAG-3')
and 5 .mu.M each of dATPO, dTTP, alpha-[.sup.32 P] dCTP and alpha-[.sup.32
P] dGTP, and 10 units of E. coli DNA polymerase I (Klenow fragment)
purchased from New England Nuclear. The reaction is allowed to proceed for
18 hours at room temperature.
EXAMPLE 5
Purification of the product by gel electrophoresis.
When the product of Example 1 or 3 is used as templates, the
electrophoretic mobility (on a 20% polyacrylamide gel) of the product is
different than the template and the short primer. The reaction mixture
(Example 4) is diluted to 40 .mu.l with loading buffer containing 8M urea,
0.05% xylene cyanol FF and 0.05 Bromophenol blue in 20 mM tris EDTA buffer
(pH 7.5). The mixture 40 .mu.l is loaded onto a 20% polyacrylamide slab
gel (0.2.times.15.5.times.29.5 cm) which has been preelectrophoresed for 1
hour at 1000 V. The gel is run for 8 hours at 1000 V in 50 mM trisborate
buffer pH 8.3. After the run autoradiographic detection is done to locate
the radioactive product band; it is excised and eluted from the gel using
1 mM EDTA solution as described by Studencki and Wallace (DNA, 3 (1984)7).
EXAMPLE 6
Purification of the product by high pressure liquid chromatography.
Both the products of Examples 1 and 3 are more hydrophobic than their
parent oligonucleotide. They can be separated from the labeled
complementary stand on a reverse phase system using acetonitrile gradient.
An identical device and reaction conditions as have been described in
Example 3 are used to separate the labeled product from the template. In
this method a part of the primer (unreacted) coelutes with the product.
This can be purified on a gel filtration system.
EXAMPLE 7
Hybridization of the product of Example 5 or 6 with DNA from .beta.-globin
gene.
The specific oligonucleotide fragment chosen for the examples (5,6) is
usable for the detection of sickle cell anemia. This particular sequence
of oligonucleotide will hybridize with DNA extracted from normal blood and
under certain conditions it will not hybridize to the DNA extracted from
the blood of a person who has sickle cell anemia disease. The extraction
of DNA from blood samples and the digestion with a restriction enzyme and
other appropriate treatment of the hybridization process have been
described by Studencki and Wallace (DNA 3 (1984) 7)). The newly labeled
material shows similar hybridization property as a .sup.32 P labeled probe
as has been described by Studencki & Wallace (DNA 3 (1984) 7)).
It will be understood that the specification and examples are illustrative
but not limitative of the present invention and that other embodiments
within the spirit and scope of the invention will suggest themselves to
those skilled in the art.
* * * * *
|
|
 |
|
 |
Description |
|
