U.S. patent application number 09/970412 was filed with the patent office on 2002-07-25 for method of sequencing.
Invention is credited to Asp, Allan, Carstenius, Peder.
Application Number | 20020098499 09/970412 |
Document ID | / |
Family ID | 20400257 |
Filed Date | 2002-07-25 |
United States Patent
Application |
20020098499 |
Kind Code |
A1 |
Asp, Allan ; et al. |
July 25, 2002 |
Method of sequencing
Abstract
A method of analyzing a sequence of a polynucleotide of
interest, comprising the steps of: a) incorporating one member of a
specific binding pair at the end of each strand of a double
stranded polynucleotide of interest, the number being of the same
type for both strands, b) immobilizing both strands of the
polynucleotide to a solid support provided with the other member of
the specific binding pair, c) annealing sequencing primers to the
immobilized strands, d) sequencing both strands by the chain
termination method. The polynucleotide of interest is preferably
amplified before or in connection with step a) and most preferably
by polymerase chain reaction extension. The invention also
comprises a kit for use in analyzing the sequence of a
polynucleotide of interest.
Inventors: |
Asp, Allan; (Uppsala,
SE) ; Carstenius, Peder; (Enskede, SE) |
Correspondence
Address: |
Amersham Pharmacia Biotech, Inc.
800 Centennial Avenue
Piscataway
NJ
08855
US
|
Family ID: |
20400257 |
Appl. No.: |
09/970412 |
Filed: |
October 3, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09970412 |
Oct 3, 2001 |
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09068783 |
Feb 22, 1999 |
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09068783 |
Feb 22, 1999 |
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PCT/SE96/01464 |
Nov 13, 1996 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 435/91.2 |
Current CPC
Class: |
C12Q 2535/101 20130101;
C12Q 2563/131 20130101; C12Q 2565/518 20130101; C12Q 2531/113
20130101; C12Q 2535/101 20130101; C12Q 1/6869 20130101; C12Q 1/6869
20130101; C12Q 1/6869 20130101 |
Class at
Publication: |
435/6 ;
435/91.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 16, 1995 |
SE |
9504099-4 |
Claims
1. A method of analyzing a sequence of a polynucleotide of
interest, comprising the steps of: a) incorporating one member of a
specific binding pair at the end of each strand of a double
stranded polynucleotide of interest, the member being of the same
type for both strands, b) immobilizing both strands of the
polynucleotide to a solid support provided with the other member of
the specific binding pair, c) annealing sequencing primers to the
immobilized strands, d) sequencing both strands by the chain
termination method.
2. A method according to claim 1, characterized in that the
polynucleotide of interest is amplified before or in connection
with step a).
3. A method according to claim 2, characterized in that said
polynucleotide is amplified by polymerase chain reaction extension
of a first and second amplification primer, one primer being
annealed to each strand of the double stranded polynucleotide,
wherein both primers comprise the member of the specific binding
pair, the member being of the same type for both primers, thereby
producing copies of both strands of the polynucleotide bonded to
said member of the specific binding pair.
4. A method according to any of the claims 1-3, characterized in
that the immobilization in step b) is made under denaturating
conditions.
5. A method according to any of the claims 1-3, characterized in
that the strands are denatured after immobilization in step b).
6. A method according to claim 1, characterized in that the
sequencing primers are differently labelled.
7. A method according to claim 6, characterized in that the labels
are different fluorescent dyes.
8. A method according to claim 1, characterized in that the solid
support is a manifold having a plurality of individual solid phase
members.
9. A method according to claim 8, characterized in that the solid
phase members are adapted for cooperation with a corresponding set
of receptacles.
10. A metod according to claim 1, characterized in that the
specific binding pair is selected from biotin--avidin,
biotin--streptavidin, cystein--thiol groups, antigen--antibody,
lectin--sugar.
11. A kit for use in analyzing the sequence of a polynucleotide of
interest comprising: (a) a solid support, (b) amplification primers
comprising one member of a specific binding pair, the member being
of the same type for both primers, (c) sequencing primers.
12. A kit according to claim 11, characterized in that the solid
support is a manifold having a plurality of individual solid phase
members and that the sequencing primers are differently
labelled.
13. The method according to any of claims 1-10 for confirmatory
sequencing e.g. DNA diagnosis, in forensic analysis, HLA typing.
Description
[0001] The present invention relates to a method of analyzing the
sequence of a polynucleotide of interest. More precisely the
invention relates to a method of sequencing both strands of the
polynucleotide whereby the strands are immobilized to a solid
support.
[0002] DNA sequencing is one of the most important technique by
which the precise order of nucleotides in a piece of DNA in the
genome of living organisms can be determined. There are two well
establised methods used: The chemical degradation method by A.
Maxam and W. Gilbert and the chain termination method by F. Sanger
and A. R. Coulson. Today the sequencing methods are routine
processes performed with automated equipment. Most automated
sequensers are based on the chain termination method and utilize
fluorescent detection.
[0003] According to the chain termination method the double
stranded polynucleotide to be analyzed is separated in a first
step, whereafter a sequencing primer is hybridized to the single
strands. The problem with this step is that the re-annealing of the
two strands compete with the primer hybridization. A method to
avoid this, which has been developed during the last years, is
solid phase sequencing. One strand of the double stranded
polynucleotide is provided with one member of a specific binding
pair. The other member of the binding pair is coated on a solid
support. This binding pair is used to bind the polynucleotide to
the support. After binding, the polynucleotide is denaturated and
the strand without attachment member is washed away, thereby
leaving the support with a single strand of polynucleotide attached
to it. Then, the sequencing primers are added and there is no
competition from re-annealing of the other strand. In this way much
more pure sequencing products are obtained. More pure products
result in better or sharper bands or signals from the detection
instruments. This is very important e.g. in detection of mutations.
With the solid phase sequencing technique it is also easy to
automate the handling of the samples.
[0004] Usually, in the solid phase technique only one of the two
strands of a double stranded molecule has been provided with a
binding member of a specific binding pair. Thereby only one of the
two strands have been captured to the solid support and used in the
sequencing reaction. It has been believed that only one of the
strands can be used, due to the problems related to working with
two complementary strands of equal size that tend to re-anneal.
[0005] For many applications it is desired to sequence both
strands, for example to obtain a double check of the sequencing
result. EP 371 437 relates to a solid phase sequencing method
which, as one embodiment, utilizes both strands of the molecule.
However, in this patent different binding members are attached to
the two strands and the strands are captured to two different solid
matrices. Thus, also with this method the risk for re-annealing has
brought about the need for separate processing of the two
strands.
[0006] The object of the present invention is to obtain an improved
method of sequencing on a solid support.
[0007] A further object of the present invention is to present a
sequencing method on a solid support by which both strands of a
double stranded polynucleotide can be sequenced at the same time on
the same solid support.
[0008] The objects of the invention are achieved by the method as
claimed in the claims. According to the invention a method of
analyzing a sequence of a polynucleotide of interest is obtained.
The method comprises the steps of:
[0009] a) incorporating one member of a specific binding pair at
the end of each strand of a double stranded polynucleotide of
interest, the member being of the same type for both strands,
[0010] b) immobilizing both strands of the polynucleotide to a
solid support provided with the other member of the specific
binding pair,
[0011] c) annealing sequencing primers to the immobilized
strands,
[0012] d) sequencing both strands by the chain termination
method.
[0013] It has been found that the solid phase technique makes it
possible to capture both strands of a polynucleotide in such an
orientation that the tendency of having them re-annealing is
avoided or largely reduced. Normally the two strands anneal in a
manner that the 5'end of one strand anneal to the 3'end of the
complementary strand. It was found that it was possible to attach a
binding member to the 5'end of each strand and to capture each
strand to the solid support by this binding member. Then the two
strands are separated by denaturation or the capture is made under
denaturating conditions. The capture of the two strands by their
5'end makes it more difficult for the two strands to "find" each
other as the orientation of the strands is "wrong" for annealing to
occur. It was surprisingly found that this "wrong" orientation was
sufficient to avoid re-annealing. The expected "rearrangement" of
the strands to re-anneal did not occur. The method according to the
invention makes it possible to sequence both strands of one double
stranded DNA at the same time on the same solid support. By using
two differently labelled sequencing primers, one for each strand,
the double amount of information is obtained from the sequencing
reaction. Hence, it is possible to reduce the number of sequencing
reactions by half, as two "different" DNA strands can be used as
templates in the same sequencing reaction.
[0014] Incorporation of-one member of the specific binding pair can
be accomplished in conventional manners.
[0015] According to a prefered embodiment of the invention the
polynucleotide of interest is amplified before or in connection
with step a) of the method. Different amplification methods can be
used such as amplification by a vector, e.g. as described in U.S.
Pat. No. 5,405,746. According to this method the member of the
specific binding pair is incorporated into the vector DNA by first
linearizing the vector with restriction enzymes. Then the binding
member is incorporated by ligation or by a DNA polymerase.
[0016] In a further prefered embodiment of the invention said
polynucleotide is amplified by polymerase chain reaction extension
or a first and second amplification primer, one primer being
annealed to each strand of the double stranded polynucleotide. Both
primers comprise the member of the specific binding pair and the
members are of the same type for both primers, i.e. only one type
of binding pair is used. In this manner copies of both strands of
the polynucleotide bonded to said member of the specific binding
pair are produced. The binding member can be incorporated into the
5'end of the primer or internally in the primer.
[0017] Polymerase chain reaction (PCR) is a common, well-known
amplification method which results in selective amplification of a
chosen part of DNA molecule. The part of the DNA molecule is
defined by a pair of primers wich are annealed to the molecule, one
primer to each strand of the double stranded molecule.
[0018] The specific binding pair can be any pair of compounds with
strong interaction between the members of the pair. Furter, it must
be possible to incorporate one of the members of the pair into
deoxynucleotides and to provide a solid support with the other
member. The interaction between the members of the pair must be
stable through the whole process. Example of such binding pairs are
biotin--avidin, biotin--streptavidin, cystein--thiol groups,
antigen--antibody, lectin--sugar.
[0019] The immobilization of the strands is performed in a
conventional manner. The denaturing step can be performed during
the immobilization or after. The strands can be separated with well
known methods such as temperature increase or NaOH addition.
[0020] The solid support used according to the invention can be any
one known in the art, e.g. magnetic or other beads, capillaries,
microtitre wells. However, preferably the solid support is a
manifold having a plurality of individual solid phase members. The
solid phase members are adapted for cooperation with a
corresponding set of receptacles, which contain the solutions for
the different reaction steps. One preferred such system is
disclosed in PCT/SE93/00929.
[0021] After the immobilization step the conditions are adjusted
for sequencing according to any well known standard protocol. The
sequencing products are labelled in a suitable manner by
introducing known labels. As such can be mentioned an isotope such
as .sup.32P or a fluorescent group. Preferably the sequencing
primers are labelled. They must be differently labelled. Most
preferably the labels are two different fluorescent dyes.
[0022] The sequencing products are separated and detected by known
means, e.g. by gel electrophoresis. If the labels are two different
fluoroscent dyes, the sequencing products can be detected for
example by a two laser instrument. Thereby the products from the
sequencing of both strands can be detected simultaneously.
[0023] The invention also comprises a kit for use in analyzing the
sequence of a polynucleotide of interest. The kit comprises:
[0024] (a) a solid support,
[0025] (b) amplification primers comprising one member of a
specific binding pair, the member being of the same type for both
primers,
[0026] (c) sequencing primers.
[0027] Preferably the solid support is a manifold having a
plurality of individual solid phase members and the sequencing
primers are differently labelled with fluorescent dyes.
[0028] The method according to the invention can be used for
determining one or several nucleotide variations, such as a
nucleotide replacement, deletion or insertion, in the
polynucleotide of interest. The method is especially suitable for
confirmatory sequencing e.g. DNA diagnosis, in forensic analysis,
HLA typing.
[0029] The invention will now be illustrated with the following
example, which however, is not intended to limit the invention.
EXAMPLE
[0030] Determination of the sequence of pUC 18 DNA.
[0031] Ten different samples of the same sequence were processed at
the same time using the AutoLoad.TM.Solid Phase sequencing kit
together with AutoLoad.TM.Heating Block Inserts from Pharmacia
Biotech.
[0032] Amplification reaction
[0033] The following components were added to a PCR tube:
[0034] 5 .mu.l of 10.times.PCR buffer
[0035] 5 .mu.l of 10.times.dNTP solution
[0036] 2 .mu.l of 5 pmole of each primer
[0037] 1-2 U (U=units) Taq polymerase from Perkin-Elmer
[0038] 5-20 ng of pUC 18 template
[0039] sterile water to obtain a total volume of 50 .mu.l
[0040] The tube was processed for 30 cycles where each cycle
comprises denaturing at 95.degree. C. for 30 sec. and primer
annealing and extension at 70.degree. C. for 120 sec. The
amplification was carried out on a GeneAmp.TM. PCR System 9600 from
Perkin-Elmer.
[0041] 10.times.PCR buffer: 100 mM Tris-HCl (pH 8.3), 15 mM
MgCl.sub.2, 500 mM KCl, 1% Tween 20.
[0042] 10.times.dNTP: 2 mM of each dNTP
[0043] primer: 5'- Biotin - GCT TCC GGC TCG TAT GTT GTG TG-3'5'-
Biatin - AAA GGG GGA TGT GCT GCA AGG CG-3'
[0044] Immobilisation of amplified DNA on solid support
[0045] As solid support was used a comb with 8 teeth coated with
streptavidin. The teeth were arranged on the comb in two rows of 4
teeth. The comb was adapted for use together with well plates. 10
well plates were used in all steps until the sequencing reactions.
One row of 4 teeth of the comb was placed in each well. For the
sequencing a 40 well plate was used and one tooth of the comb was
placed in each well. This is in accordance with FIG. 7 in the above
mentioned PCT/SE93/00929
[0046] 80 .mu.l of a binding/washing solution consisting of 2M
NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA were added to each well of
a 10 well plate. 40-50 .mu.l of the reaction mixture from the PCR
reaction were added to each well. The streptavidin coated combs
were placed in the wells and were moved up and down gently to
obtain mixing. Then the solution was incubated 1 hour at room
temperature to ensure complete immobilization of the biotinylated
PCR products on the combs. The immobilized products were washed in
the same binding/washing solution as above.
[0047] The immobilized DNA was denatured by placing the combs in
the wells of a 10 well plate and incubating the combs for 5
minutes. Each well contained 100 .mu.l 0.1M NaOH solution. The
denatured DNA was washed, first with 1.times. TE buffer (10 mM
Tris-HCl, 1 mM EDTA (pH 8)) and then with sterile water.
[0048] Sequencing of immobilized DNA
[0049] Primer annealing:
[0050] The following components were added to each well of a 10
well plate, whereby one well is required for each PCR product being
sequenced:
[0051] 12 .mu.l annealing buffer (1M Tris-HCl (pH 7.6), 100 mM
MgCl.sub.2)
[0052] 4 pmoles (4 .mu.l)primer 1 (M13 "Universal" primer),
labelled with Cy-5.TM. (red indocarbocyanine fluorophore)
[0053] 4 pmoles (4 .mu.l) primer 2 (M13 "Reverse" primer) labelled
with a "blue" label (fluorescein)
[0054] 100 .mu.l sterile water
[0055] The combs with immobilized DNA were placed in the wells
which were heated to 65.degree. C. for 10 minutes. Then the wells
were cooled to room temperature for at least ten minutes.
[0056] Sequencing reactions:
[0057] The following reagents were mixed to four different mixture,
the A, C, G and T mixture:
[0058] 30 .mu.l termination mixture of A, C, G or T
respectively
[0059] 20 .mu.l annealing buffer
[0060] 10 .mu.l extension buffer
[0061] 120 .mu.l sterile water
[0062] 10 .mu.l diluted T7 DNA polymerase
[0063] 10 .mu.l dimethyl sulphoxide
[0064] Each termination mixture consists of: 1 mM dATP, 1 mM dCTP,
1 mM dTTP, 1 mM c7dGTP, 50 mM NaCl and 40 mM Tris-HCl (pH 7.6). The
A termination mixture further consists of 5 .mu.m ddATP, the C
mixture of 5 .mu.m ddCTP, the G mixture of 5 .mu.m of ddGTP and the
T mixture of 5 .mu.m of ddTTP.
[0065] Annealing buffer used was: 1M Tris-HCl (pH 7.6) and 100 mM
MgCl.sub.2.
[0066] Extension buffer: 304 mM citric acid, 324 mM DTT
(dithiothreitol) and 40 mM MnCl.sub.2 (pH 7.5)
[0067] T7 DNA polymerase: 8 units/.mu.l in 25 mM Tris-HCl (pH 7.5)
, 0.25M NaCl, 5 mM DTT and 50% glycerol. This polymerase solution
was diluted with a buffer consisting of Tris-HCl (pH 7.5), 5 mM
DTT, 100 .mu.g/ml BSA (bovine serum albumine) and 5% glycerol to a
concentration of 2.0 units/.mu.l. 4 .mu.l of this diluted stock
solution will be required for each template (set of four
reactions).
[0068] 19 .mu.l of each of A, C, T and G mixture were added to
respective well in the 40 well sequencing reaction plate. The plate
was heated to 37.degree. C. whereafter the combs were added. The
combs were incubated at 37.degree. C. for 5 minutes and then
cooled.
[0069] The separation and detection of the sequencing products was
done on a modified ALF.TM. DNA Sequencer from Pharmacia Biotech.
The sequencer comprises a electrophoresis module for separation and
a double laser for detection. Before loading the samples onto the
sequencer, 10 .mu.l Stop solution was added to each gel well in the
sequencer. The temperature was raised to 50.degree. C. and then the
comps were loaded into their respective wells. After 10 minutes of
incubation, the combs were removed and the electrophoresis
started.
[0070] The invention is chematically illustrated in FIG. 1. In FIG.
1. a) Biotin (B1 and B2 in FIG. 1) has been incorporated at the end
of each strand of a double stranded polynucleotide. In FIG. 1 b)
the biotinylated strands have been immobilized to a solid support
and in c) a blue and a red sequencing primer respectively, have
been annealed to the strands.
[0071] The result of the example is shown in FIG. 2. In FIG. 2 the
graphs obtained with the red label are shown in the lower plot and
the graphs obtained with the blue label in the top plot. If the
lower plot is read from the right to the left and the top one from
the left to the right, it can be seen that the two plots and thus
the two strands complement each other. The first nucleotide of the
lower strand is an A which complements the T which is the first
nucleotide in the upper strand. The next nucleotide is a G which
complements C and C G etc.
Sequence CWU 1
1
2 1 23 DNA Artificial Sequence Description of Artificial Sequence
synthetic oligonucleotide 1 gcttccggct cgtatgttgt gtg 23 2 23 DNA
Artificial Sequence Description of Artificial Sequence synthetic
oligonucleotide 2 aaagggggat gtgctgcaag gcg 23
* * * * *