U.S. patent number 3,730,844 [Application Number 05/175,756] was granted by the patent office on 1973-05-01 for polynucleotide analysis.
This patent grant is currently assigned to Purdue Research Foundation. Invention is credited to Peter Thomas Gilham, Herbert Lee Weith.
United States Patent |
3,730,844 |
Gilham , et al. |
May 1, 1973 |
POLYNUCLEOTIDE ANALYSIS
Abstract
Sequential analysis of a polynucleotide to determine the
particular order of nucleoside units therein can be conveniently
carried out by adsorbing a polynucleotide on a strongly basic
anion-exchange material, oxidizing the terminal nucleoside of the
polynucleotide with a periodate, removing any excess periodate by
reaction with L-rhamnose, treating the adsorbed polynucleotide with
an amine to remove the terminal nucleoside residue from the
polynucleotide molecule and with a phosphatase to remove the
resulting terminal phosphate group from the remaining
polynucleotide molecule, separating the so-produced nucleoside
residue from the adsorbed polynucleotide for subsequent
identification and then repeating the above procedure for each
remaining nucleoside unit of the polynucleotide.
Inventors: |
Gilham; Peter Thomas (West
Lafayette, IN), Weith; Herbert Lee (West Lafayette, IN) |
Assignee: |
Purdue Research Foundation
(Lafayette, IN)
|
Family
ID: |
22641511 |
Appl.
No.: |
05/175,756 |
Filed: |
August 27, 1971 |
Current U.S.
Class: |
435/6.12;
435/6.1; 536/25.4; 435/21 |
Current CPC
Class: |
C12Q
1/6834 (20130101); C12Q 1/42 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12Q 1/42 (20060101); G01n
031/14 () |
Field of
Search: |
;195/103.5,28N |
Other References
method in Enzymology, Volume XII, Nucleic Acids Part B Pages
224-235 (1968)..
|
Primary Examiner: Tanenholtz; Alvin E.
Claims
What is claimed is:
1. A process for the sequential analysis of a polynucleotide which
comprises (1) adsorbing on a strongly-basic anion-exchange material
a polynucleotide having its terminal 3' phosphate group previously
removed, (2) treating the adsorbed polynucleotide with a periodate
to oxidize the unsubstituted cis-hydroxyl groups of the terminal
nucleoside unit of the polynucleotide to dialdehyde groups, (3)
adding L-rhamnose to react with and remove any remaining periodate
material, (4) treating the adsorbed polynucleotide with an amine to
remove the terminal nucleoside unit from the polynucleotide
molecule and at substantially the same time with a phosphatase to
remove the resulting terminal 3' phosphate group from the remaining
polynucleotide molecule, (5) separating the so-produced nucleoside
residue from the adsorbed polynucleotide for subsequent
identification, and then repeating the above steps (2) through (5)
inclusive for each remaining nucleoside unit of the
polynucleotide.
2. A process according to claim 1 wherein steps (2), (3) and (5)
take place at about 1.degree. C. and step (4) takes place at about
45.degree. C.
3. A process according to claim 1 wherein prior to steps (2), (4)
and (5) the concentrations of anions, other than those of the
polynucleotide, in the liquid in contact with the anion-exchange
material are reduced to a level such that they do not displace the
polynucleotide from being adsorbed by the anion-exchange
material.
4. A process according to claim 1 wherein the strongly-basic
anion-exchange material is a polystyrene cross-linked with
divinylbenzene and containing quaternary ammonium reactive groups.
Description
BACKGROUND OF THE INVENTION
Polynucleotides or polyribonucleotides are known to be long chain
polymers containing various individual nucleoside or ribonucleoside
units. Each nucleoside unit consists of a ribose containing a
purine or pyrimidine substituent. The ribose portions of adjacent
nucleosides are linked through phosphate groups. It is often of
importance in biochemical and medical research to know the specific
order in which the nucleoside units are attached in the formation
of the polynucleotide molecule. Various techniques have been
proposed in the prior art for degradation of the polynucleotide
molecule into separate nucleoside fragments which can then be
individually analyzed to determine the purine or pyrimidine bases
from which they were formed. The final desired analytical result is
the particular sequence of bases in the polynucleotide chain.
One technique proposed for analysis of a polynucleotide involved
exonucleolytic enzymes which allegedly would split off the terminal
nucleoside units one at a time for subsequent analysis. This
enzymatic technique was not successful because the proposed enzymes
had variable and non-reproducible activity and produced inaccurate
results.
A stepwise chemical and enzymatic degradation procedure was then
proposed. This process involved reaction with a phosphatase to
remove the terminal 3' phosphate group of the polynucleotide,
oxidation of the unsubstituted cis-hydroxyl groups of the terminal
nucleoside unit to dialdehyde groups, followed by alkaline
catalyzed elimination of the terminal nucleoside fragment. The
so-produced fragment was then identified for its purine or
pyrimidine substituent. This procedure was then repeated for each
nucleoside unit of the polynucleotide molecule. This proposed
procedure had several disadvantages. First, there was no simple and
efficient means for separating the liberated nucleoside fragment
since all the reaction components and products were in solution.
Second, great care must be taken to avoid the simultaneous presence
in the reaction mixture of the phosphatase, periodate and alkali.
Otherwise, the cleavage of the nucleoside fragments might occur in
an uncontrolled manner to produce erroneous results.
A process improvement was then suggested to employ ion exchange
chromatography to separate the liberated nucleoside residue from
the remaining polynucleotide molecule after each degradation cycle.
This was successful but had the disadvantages of being quite time
consuming and of sustaining significant material losses. It could
therefore be used only for a relatively few degradation cycles and
thus could not be used for analysis of more complex polynucleotide
molecules.
Attempts to precipitate the liberated nucleoside residues in order
to separate them from the remaining polynucleotide molecule have
also been unsuccessful due to excessive manipulation and consequent
losses of material.
It is an object of the present invention to provide an accurate and
convenient process for the sequential degradation of a
polynucleotide into distinct reproducible nucleoside fragments
which can subsequently be identified as to their purine or
pyrimidine bases.
SUMMARY OF THE INVENTION
In accordance with the present invention, a process is provided for
the sequential analysis of a polynucleotide which comprises (1)
adsorbing on a strongly-basic anion-exchange material a
polynucleotide having its terminal 3' phosphate group previously
removed, (2) treating the adsorbed polynucleotide with a periodate
to oxidize the unsubstituted cis-hydroxyl groups of the terminal
nucleoside unit of the polynucleotide to dialdehyde groups, (3)
adding L-rhamnose to react with and remove any remaining periodate
material, (4) treating the adsorbed polynucleotide with an amine to
remove the terminal nucleoside unit from the polynucleotide
molecule and at substantially the same time with a phosphatase to
remove the resulting terminal 3' phosphate group from the remaining
polynucleotide molecule, (5) separating the so-produced nucleoside
residue from the adsorbed polynucleotide for subsequent
identification, and then repeating the above steps (2) through (5)
inclusive for each remaining nucleoside unit of the
polynucleotide.
DESCRIPTION OF THE INVENTION
The polynucleotides useful as raw materials in the sequential
analysis process of the present invention are well known
polyribonucleotide compounds which occur naturally in biological
materials or can be produced synthetically. In order to be
initially useful in this process the polynucleotide must have its
terminal 3' phosphate group removed. This is conveniently
accomplished through the known use of an alkaline phosphatase.
The strongly basic anion-exchange materials useful in the present
invention are well-known and are commercially available. They are
prepared, for example, by suspension polymerization of styrene and
divinylbenzene. The resulting polymer beads are reacted with
chloromethyl ether, in the presence of aluminum chloride or zinc
chloride catalyst, to introduce --CH.sub.2 Cl groups on the benzene
rings of the polymer. This product is then aminated with
trimethylamine, for example, to form a highly ionized quaternary
ammonium group on the benzene rings.
Strongly basic anion-exchange resins having quaternary ammonium
reactive groups are sold under the following illustrative
tradenames by the indicated suppliers.
Tradename Supplier Dowex 1 Dow Chemical Co. Dowex 2 Dow Chemical
Co. Dowex 21 K Dow Chemical Co. Amberlite IRA-400 Rohm and Haas Co.
Amberlite CG-400 Rohm and Haas Co. Amberlite IRA-401 Rohm and Haas
Co. Nalcite SBR National Aluminate Co. Nalcite SBR-P National
Aluminate Co. Duolite A-101 D Diamond Alkali Co. Duolite A-102 D
Diamond Alkali Co. Permutit S-100 The Permutit Co. Permutit S-200
The Permutit Co.
The preferred anion-exchange material for use in the present
invention is Dowex 1 .times. 2, which is a Dowex 1 material
consisting of a polystyrene cross-linked with 2 percent
divinylbenzene and also containing quaternary ammonium reactive
groups. This material has desired chemical stability and sufficient
anion-exchange capacity over the wide range of pH values
encountered in this process.
The periodate compounds useful to oxidize the cis-hydroxyl groups
of the dephosphorylated terminal nucleoside unit of the
polynucleotide are well-known, and the general reaction conditions
are known.
The use of hydroxyl-containing materials, such as ethylene glycol
and butane-2, 3-diol,to react with excess periodate is also known.
It is preferred in the process of the present invention to employ
L-rhamnose since this material has been found to be most efficient
and is the fastest reacting substance for this purpose. This tends
to reduce the overall process time, which is an advantage over the
prior art.
The use of alkaline materials, such as amines, to degrade the
polynucleotide by removal of the terminal nucleoside fragment is
known in the art. It is preferred in the process of the present
invention to employ a mixture of cyclohexylamine and
N,N,N',N'-tetramethylglycinamide-HCl since this mixture provides
improved pH control at the desired level of pH 8.5 during this step
of the overall process.
While the temperature conditions under which this process is
carried out are not narrowly critical, it is preferred that the
reaction of the polynucleotide with the periodate, the treatment
with the L-rhamnose and the separation of the degraded nucleoside
fragment from the adsorbed polynucleotide be carried out at about
1.degree. C. and the amine reaction with the polynucleotide to
degrade and remove the terminal nucleoside fragment be carried out
at about 45.degree. C.
The principal point of technical advancement of the present
invention resides in the adsorption of the polynucleotide on an
insoluble support, reacting various materials with this
insolubilized form of polynucleotide and easily separating the
soluble degraded nucleoside fragments from the insolubilized
remaining portion of the polynucleotide. It is important,
therefore, at the time that reaction products are to be separated
from the polynucleotide that all of the remaining polynucleotide be
adsorbed by the anion-exchange material. This is accomplished by
dilution of the liquid in contact with the anion-exchange material
to the point that the concentrations of anions, other than those of
the polynucleotide, are reduced to a level such that they do not
displace the polynucleotide being adsorbed by the anion-exchange
material. The specific conditions under which a polynucleotide is
released from the anion-exchange material and readsorbed by it are
dependent on the size of the polynucleotide molecule. For example,
a polynucleotide having ten nucleoside units is released from the
anion-exchange material when the competitive anion concentration
exceeds about 1 molar. Such a polynucleotide is completely
readsorbed when the displacing anion concentration is reduced by
dilution to about 0.1 molar. A polynucleotide containing only two
nucleoside units is released when the competitive anion
concentration exceeds about 0.4 molar and is completely readsorbed
when the competitive anion concentration is below about 0.05
molar.
When the nucleoside fragment is separated from the polynucleotide,
it can be analyzed for its purine or pyrimidine base by well-known
methods. For example, the effluent from the degradation cycle
containing the terminal nucleoside unit, amine and phosphatase is
evaporated to dryness. Formic acid is added, and the resulting
reaction mixture is heated in an autoclave. This acid treatment
converts the terminal nucleoside residue into free purine or
pyrimidine base which is then identified by anion exchange
chromatography.
The process of the present invention is described in additional
detail in the following illustrative example.
EXAMPLE
A 0.1 ml portion of Dowex 1 .times. 2 anion-exchange resin in the
chloride form and having a particle size of minus 400 mesh was
placed in a glass tube and positioned by plugs of glass wool. The
resulting resin bed was washed with a buffer mixture of 0.5 molar
sodium chloride and 0.01 molar tris (hydroxymethyl) amino-methane
having a pH of 7.5 and then with cold distilled water to remove
excess buffer solution. The temperature of the resin bed was
maintained at about 1.degree. C. by means of a water bath
surrounding the resin bed.
The polynucleotide to be analyzed was then treated with alkaline
phosphatase to remove the terminal 3' phosphate group. An aqueous
solution containing about 100 nanomoles of the thus
dephosphorylated polynucleotide was passed through the above resin
bed and recirculated through the bed several times by means of a
recirculating pump and associated tubing. Most of the
polynucleotide was adsorbed by the resin. Any unadsorbed
polynucleotide was then removed from the resin by further washing
with distilled water. A 0.5 ml. portion of 0.2 molar sodium
metaperiodate solution was then passed through the bed and
recirculated through the bed at 1.degree. C. for about 15 min. This
periodate solution oxidized the cis-hydroxyl groups on the terminal
nucleoside unit to dialdehyde groups and, because of its ionic
effect, also displaced the polynucleotide from the resin. The
solution being recirculated through the resin thus contained
polynucleotide. A 0.5 ml. portion of 1 molar L-rhamnose solution
was then added to the circulating solution, and the recirculation
through the bed was continued for 5 min. during which time the
L-rhamnose destroyed any previously unreacted periodate. A 4.6 ml.
portion of cold distilled water was then added to the reaction
vessel so as to dilute the resulting iodate ion concentration to
about 0.017 molar. Recirculation of the total liquid mixture was
continued for 10 minutes to allow the polynucleotide to become
readsorbed by the resin bed. The liquids were then drained from the
resin bed, and the resin bed was washed with 1 ml. of cold
distilled water. A 0.1 ml. portion of bacterial alkaline
phosphatase was then added to the resin bed followed by 0.1 ml. of
an amine solution containing 1 molar cyclohexylamine and 2 molar
N,N,N',N' -tetramethylglycinamide-HCl. An additional 0.1 ml. of
amine solution was added and the liquids were circulated through
the resin bed at 45.degree. C. for 2 hours. This amine-phosphatase
mixture removed the terminal nucleoside unit from the remainder of
the polynucleotide molecule and also removed the so-generated
terminal 3' phosphate group. This solution, because of its ionic
effect, also displaced the polynucleotide from the resin bed. A 5.0
ml. portion of distilled water was then added to the reaction
mixture so as to dilute the amine concentration to about 0.04
molar. The temperature in the resin bed was reduced to about
1.degree. C. and the above liquid mixture was recirculated through
the resin bed at 1.degree. C. for 15 min. to allow the
polynucleotide (minus its original terminal nucleoside unit) to
become readsorbed by the resin bed. The diluted
amine-phosphatase-terminal nucleoside fragment mixture was then
drained from the resin bed into a screw cap test tube. The reaction
vessel and the resin bed were then washed with 1 ml. of cold
distilled water which was also drained into the above test tube.
The total time for the above periodate oxidation, terminal
nucleoside elimination and dephosphorylation was about 200 min. The
resin bed containing adsorbed polynucleotide was then treated again
by the above reaction steps to eliminate a further terminal
nucleoside unit. This procedure was repeated until all the
neucleoside units of the polynucleotide were separately
removed.
Each of the combined effluents from a single degradation cycle
having an average volume of about 8 ml. was individually heated at
100.degree. C. in a sealed tube for two hours. The resulting free
purine or pyrimidine base in each test tube was individually
analyzed by anion exchange chromatography.
This above procedure was employed to confirm the sequence of
nucleoside units in a polynucleotide of known sequential
composition and has been employed to determine the sequence of
nucleoside units in polynucleotides of previously unknown
sequential composition.
The practice of the process of the present invention enables
accurate analyses to be carried out in a shorter amount of time and
with more complex polynucleotides than the practice of the prior
art processes. The simplicity of operation of this process also
lends itself to possible automation of the apparatus for carrying
out the process.
* * * * *