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Description  |
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The invention is related to a method of sequencing DNA using a solid phase
bound DNA template.
The increased interest in large scale sequencing projects, such as
proposals to sequence the entire human genome (Smith et al (1987)),
necessitates technical improvements to enable megabase sequencing.
Sequencing can be divided into the following five operations, which all
must be automated to enable large scale projects: (i) template
preparation, (ii) sequence reactions, (iii) electrophoresis, (iv)
detection of specific fragments and (v) data storage and analysis. Except
of data storage and analysis, most sequencing is at present carried out
manually, causing considerable investments in operator time.
Recently, many technical improvements have been reported, although the
major contributions concern the data evaluation, i.e. computer software. A
filter method to perpare single stranded phage DNA has been described
(Kristnesen et al (1987)), which may be developed into an automated
procedure. Attempts to develop automated sequencing reactions by a
centrifugal reagent handling device have also been described (Martin et al
(1985)) as well as image processing programs for the detection of the
bands on the radiograms (Elder et al (1985)). However, the most common
approach has been to automate techniques with the aid of robots. Using
such a strategy, systems for high-speed sequencing (Wada et al 1983)) and
DNA template preparations (De Bonville et al 1987)) have been introduced.
A novel approach to automize the electrophoresis step has been described by
several groups (Smith et al (1986), Ansorge et al (1987) and Prober et al
(1987)) taking advantage of fluoresence instead of isotopes for labelling
the DNA fragments. With these systems on-line detection can be achieved,
which makes it possible to combine the three operations electrophoresis,
detection and data handling into a single automated station. Such systems
are therefore likely to be included in megabase sequencing strategies.
To obtain a completely automated sequencing protocol, it is therefore
essential to also develop suitable automated methods for the first two
operations (template preparation and sequencing reactions). For the latter
operation a strategy involving solid phase techniques would facilitate
automated handling of liquids in microliter quantities, which would be
suitable for automated protocols.
According to the present invention we provide a method of sequencing target
DNA in which said DNA is provided in double stranded form immobilized on a
solid support via one terminus of one of the two strands thereof and is
then subjected to strand separation whereby the unattached strand is
removed prior to sequencing the immobilized strand.
Solid phase methods have proven to be very useful in molecular biology, in
areas such as peptide synthesis, peptide sequencing and DNA synthesis. A
large number of instruments are commercially available utilizing this
technique. The advantage with a solid phase approach is usually a
combination of good yields, reproducable reactions and easy automation due
to ease of separation of the solid phase from the reaction solution.
At present there are, however, few reports on solid phase approaches to
handling manipulatations of cloned DNA sequences for applications such as
DNA sequencing reactions. DNA sequencing of oligonucleotides on
anion-exchange supports (Rosenthal et al, 1985) has been described, but
most attempts to automate DNA sequencing have been focused on the use of
laboratory robots (Martin et al, 1985, and Wada et al, 1987).
H. Delius et al (Nucleic Acids Research, Vol 13, No 15, 1985 p 5457) has
described immobilisation of double stranded DNA and strand specific
elution but only in the context of electron microscopic analysis of
heteroduplexes.
In the preferred method of the present invention a desired DNA sequence is
selectively incorporated into a double stranded plasmid or phage vector. A
functional group with affinity for a certain substance is incorporated
into the desired strand of the vector DNA. This substance is bound to a
solid phase and when the vector is brought into contact with the solid
phase it is immobilized via interaction between the functional group and
the substance. The vector is then subjected to melting and the non-bound
DNA strand is thereafter eluted under suitable conditions.
The double stranded DNA can be any vector, such as a plasmid or a phage,
that can provide suitable cloning sites for incorporation of target DNA
and also restriction sites for linearization. Two plasmid vectors
especially designed and constructed for use according to the present
invention are plasmids pRIT27 and pRIT28, described in the experimental
part. However, phage vectors such as Lambda or cosmid vectors can also be
used.
The desired DNA sequence could vary considerably in length, from a few base
pairs up to a least 30-40 thousand base pairs, depending on the
application. The DNA sequence is selectively incorporated into the vector
using recombinant DNA techniques known per se.
The functional group could be any compound which can be incorporated into
deoxynucleotides and which has a strong interaction to a substance which
can be immobilized on a solid-support. The interaction between the two
components must be stable through the whole procedure. Examples of such
groups are biotin - avidin, biotin - streptavidin, and cystein - thiol
groups.
Incorporation of the functional group A into the vector DNA must be
performed in such a way that the functional site is not affected. One
example of such a functional group for binding is 11-biotin-dUTP. The
vector must be linearized, and this can be done by suitable restriction
enzymes, such as BstEII, BglII (plasmid pRIT27) or NotI (pRIT28). Using
lambda or cosmid vectors, it is possible to linearized the vector with a
phage specific enzyme which recognizes and cleaves the cos-site, thus
avoiding restriction enzymes.
The incorporation of the functional group can be accomplished by a DNA
polymerase, such as Klenow, T7 or reverse transcriptase (Pharmacia,
Sweden), if a 5'-protruding end exists after linearisation. It is also
possible to incorporate the functional group into the vector by ligation
with a suitable oligonucleotide synthesized with the functional group.
The immobilization process may be performed in a conventional manner,
either batch-wise with the substance-coupled carrier slurried in a
suitable medium or on a column of the activated carrier. Any conventional
carrier material (such as beads e.g. Sepharose beads, (Pharmacia,
Sweden)), filters, capillaries, or plastic dipsticks (e.g. polystyrene
strips) and microtitre wells to which the substance can be sufficiently
coupled for the present purposes, may be used. The methods for coupling or
immobilizing a functional group to such carrier material are well-known
and need not be described in any detail herein. It is also possible to use
adsorption of the substance to surfaces of microtiter wells as a means for
coating.
Release or melting of the non-bound DNA strand from the carrier material
may be effected by conventional methods, such as 0.15M NaOH or temperature
increase. The choice of melting conditions must, of course, be made with
regard to the particular functional group-substance interaction as well as
the choice of carrier material.
A valuable initial step in method of the present invention is thus to
provide an immobilized single stranded recombinant DNA fragment suitable
for DNA sequencing. An example of such a procedure is schematically
outline in FIG. 1.
Briefly, the target DNA is cloned into the multi-linker region of the
sequencing vector. The plasmid is linearised with a restriction enzyme and
the protrusions are filled in using deoxynucleotide(s) with at least one
of them derivatized to contain the functional group. After restriction
with a second enzyme, the mixture is contacted with a solid support
containing a substance with affinity for the functional group. This leads
to directed immobilization of the DNA fragments containing the functional
group. Single stranded DNA is obtained by melting the strands, either by
alkali or heat treatment, and simultaneous elution of the non-functional
strand. A general sequencing primer is annealed to the resulting
immobilized single stranded template and the sequencing reaction is
performed under standard conditions (Sanger et al (1977)). The extended
oligonucleotides can be labelled using different strategies, most notably
isotopes or fluoresence which are incorporated either during the extension
or as a labelled primer. The newly synthesized labelled oligonucleotides
are eluted by another melting step leaving the template available for the
next sequencing reaction. The annealing and extension is repeated to
obtain specific fragments for all four nucleotides and the four samples
are loaded on a sequencing gel.
Another useful element in the invention is the provision of templates for
sequencing using biotinylated oligonucleotide and partial restriction
enzyme cleavage reactions to obtain DNA fragments of different lengths
with a biotin in one end of the fragment and an oligonucleotide
complimentary to a sequencing primer in the other end. An example on such
procedure is schematically outlined in FIG. 2.
Briefly, the target DNA is cloned into the multi-linker region of the
sequencing vector. The plasmid is linearized with SfiI and partially
cleaved with Sau3A to yield fragments of different lengths. Two general
oligonucleotides sfiI and Sau3A are ligated to the DNA fragments. The
oligonucleotide that ligates to one of the SfiI recognized protruding ends
carries the functional group and the oligonucleotide that ligates to the
Sau3A protruding ends contains a primer annealing sequence. The mixture is
separated by agarose or polyacrylamide gel electrophoresis and by using
radioactive, fluorescent or alternative methods, fragments containing the
SfiI oligonucleotide are visualized. The bands represent DNA fragments
containing in one end of the oligonucleotide the functional group and in
the other end the Sau3A oligonucleotide incorporated into a specific Sau3A
site. After collecting the specific bands separately from the gel and
eluting these by methods known per se, the DNA is immobilized by contact
with a solid support containing a substance with affinity for the
functional group. The sequencing is therefore carried out as described
above (FIG. 1) except that a general sequencing primer (GSPIII) is used
which is complimentary to the Sau3A oligonucleotide incorporated to the
3'-end of the immobilized fragment.
The advantage with this method is that large inserts (greater than 2000
base pairs) can be sequenced in a direct way without the need for
subcloning of smaller framgments. It is of course possible to compliment
the SfiI/Sau3A system with other enzymes such as TaqI, MspI, HpaII etc.
The immobilized single stranded DNA for sequencing may also be produced by
the polymerase chain reaction (PCR) technique whereby relatively small
amounts of the DNA to be sequenced, for example genomic DNA, can be
greatly amplified enzymically and according to a modification, also
immobilized an a solid support. In the PCR technique, two oligonucleotide
primers are selected which hybridise to respective sequences at or near
the 5'-ends of the coding and non-coding strands of the DNA to be
amplified; after annealing to hybridise the primers to the target DNA,
polymerisation is then effected using a suitable polymerase to produce a
copy of each of the coding and non-coding strands incorporating the
primers whereupon strand separation is effected, eg.g by conventional
melting for example at 90.degree. C. If an excess of the primer
oligonucleotides is included in the medium as well as the four nucleotides
required for synthesis, the separated new strands together with the
original strands can serve as templates for a further cycle of annealing,
polymerisation and strand separation. It will be seen that if this
procedure is continued through a number of repeated cycles, the target DNA
will be amplified exponentially while other DNA present will largely be
unaffected. Recently, a thermophilic polymerase has become available, Taq
1, which can withstand the melting temperature needed for strand
separation, thus. avoiding the need to add polymerase at each repeat of
the cycle as when using the Klenow polymerase used originally in PCR.
If one of the oligonucleotide primers is attached to a solid support such
as a particle or, more preferably, carries means permitting attachment to
a solid support such as biotin, the amplified DNA will be produced with
means for immobilisation. Thus, the PCR technique can produce directly
immobilized single stranded DNA ready for sequencing and may produce this
directly from a bacterial colony by a method which is easy to automate and
does not involve restriction cleavages and plasmid purification. A
particular advantage of the use of a solid support in any of the reactions
here concerned is the ease of separation from the reaction medium. Thus,
in the PCR stage, the reaction medium can be readily removed by washing
and a different polymerase introduced in an optimal buffer to begin the
sequencing stage, e.g. a conventional sequencing polymerase such as T7.
Furthermore, the optimal concentrations of nucleotides and
dideoxynucleotides can be maintained for sequencing by the Sanger method,
independently of the concentrations used in the PCR step. The possiblity
of rigorous washing of the immobilized DNA provides more reproducible
results in the sequencing stage. Furthermore, the so-called `walking
primer` technique is facilitated in Sanger sequencing whereby a primer can
be used to sequence the first 500 base pairs of a long DNA molecule and
after washing, the unchanged immobilized DNA is annealed to a primer
initiating sequencing of the next 500 base pairs (using sequence
information from the first stage), this procedure being continued until
the whole DNA molecule has been sequenced.
The invention will in the following be further illustrated by non-limiting
examples with reference to the appended drawings wherein:
FIG. 1 is a schematic drawing of the basic concept of the solid-phase
sequencing using the biotin-avidin system. Note that depending on the
choice of enzymes A and B, both fragments will or will not be immobilized.
FIG. 2 is a schematic drawing of the basic concept to use a biotinylated
oligonucleotide to sequence fragments obtained after partial cleavage with
a restriction enzyme such as Sau3A.
FIG. 3 shows the sequencing vector, pRIT27. The nucleotide sequence and the
deduced amino acid sequence in the multi-linker region is shown as well as
the sequence of the synthetic linkers inserted in the flanking regions.
Abbreviations: bla, beta-lactamase gene; ori, origin of replication; fl,
origin of replication of phage fl; lacZ', part of the beta-galactosidase
gene.
FIG. 4 shows the result of immobilization of biotinylized, double stranded
pRIT27, end-labelled with .sup.32 P. The amount of label bound to 1 ul of
avidin agarose after 30 minutes of incubation at room temperature is
shown.
FIG. 5 shows the autoradiographs of sequencing gels with samples obtained
by solid-phase sequencing. A; labelling using .sup.35 S-dATP during the
extension. B; labelling using .sup.32 P end-labelled sequencing primer.
Also shown is the expected sequence.
FIG. 6 shows the sequencing vector, pRIT28. The nucleotide sequence of the
synthetic linkers inserted in the flanking regions a-e are shown.
Abbreviations are as in FIG. 3.
Specific embodiments of the invention will now be described in detail.
MATERIALS AND METHODS
Enzymes were obtained from Parmacia Sweden, and were used according to the
suppliers recommendations. DNA manipulations were according to standard
procedures (Maniatis, 1982). 11-bio-dUTP was obtained from Bethesda Res.
Labs. (US) and avidin agarose was obtained from Sigma Chemicals.
EXAMPLE 1
Construction of Plasmid pRIT27
Plasmid pTZ18 (Pharmacia AB, Sweden)) was partially digested with BglI and
the synthetic oligonucleotide 5'-CCATGACAATGGAGTGCTGGTTACCGATATCGAA-3'
(and its complementary sequnce) was inserted. This synthetic fragment
contains BstXI, BstEII and EcoRV recognition sequences. The BglI
recognition sequence, used for the insertion, was destroyed
simultaneously. The reading frame was changed in the last part of the
lacz'-gene but the colour of the colonies remained blue, if E,coli strain
RRI 15 (Maniatis et al (1982)) IPTG/X-gal, selection was used (Maniatis et
al (1982)). This construction, designated pSS1, was digested with EcoRI
and HindIII, and a synthetic oligonucleotide
5'-AATTCGGCCAGCACGGCCGGCTCAGGTGACCA-3' was inserted. The EcoRI and HindIII
sites were thus destroyed and a sequence of SfiI, EcoRI, PstI, HindIII,
SfiI sites were created. This insertion changed the colour of the colonies
from blue to white, due to fram-shift in the lac Z'-gene. The new EcoRI
and HindIII recognition sequences were used to insert a mp 8 multi-linker,
restoring the correct frame in the lac Z'-gene giving back blue colonies.
Thereafter the PvuII site upstream of the lac Z'-gene was converted into a
BglII site, by insertion of a linker 5'-CAGATCTG-3' (Kabigen, Sweden). The
resulting plasmid was denoted pRIT27. (FIG. 3.)
Immobilization of Biotinylated Double Stranded DNA
Plasmid pRIT27 containing an insert derived from the multi-linker region of
M13 mp 18, was digested with BstEII and EcoRV. The 5' protrusions were
filled in with Klenow polymerase (Maniatis et al (1982)) using 11-bio-dUTP
and appropriate dNTP's. The material was purified by passing it through a
G-50 column, followed by ethanol precipitation. After redissolving in TE
(10 mm TRIS pH 7.5, 1 mM EDTA) the plasmid was digested with EcoRI. This
biotynlated double stranded DNA was mixed with avidin agarose gel,
prepared by washing with 1M NaCl and TE. Approximately 1 ug of plasmid
(treated as described above) was used per ul avidin agarose gel for the
immobilization. The mixture was shaken gently at room temperature for one
hour.
The capacity of the avidin agarose was determined by a saturation
experiment. Avidin agarose was mixed with increasing amounts of labelled
and biotinylated plasmid DNA and the amount of immobilized labelled
material was determined. The result (FIG. 4) demonstrates that several ug
of DNA can be bound to each ul of avid agarose, suggesting that the
capacity of the matrix is sufficiently high to allow sequencing reactions
in a reasonable scale.
Sequencing Reactions Using Immobilized Template DNA
The immobilized biotinylated double stranded DNA was converted into single
stranded form by incubation at 37.degree. C. with 0.15M NaOH for 15
minutes. The avidin agarose gel, with immobilized template DNA was
subsequently washed with 0.15M NaOH and water. Sequencing reactions were
performed using both .sup.35 S labelled dATP and .sup.32 P end labelled
primer. In both case of 1 ug of the plasmid immobilized on 1 ul avidin
agarose gel were mixed with equimolar amounts of the primer in a buffer,
containing 10 mM Tris HCl (pH 7.5) 10 mM MgCl.sub.2, 100 ug/ml BSA and 100
mM NaCl at a total volume of 10 ul. The mixture was incubated for 1 h at
60.degree. C. and allowed to cool to room temperature. The supernatant was
removed and the above described buffer, with addition of 1 ul BSA (1
mg/ml), was added, together with 1 ul Klenow polymerase and 5 ul of the
respective nucleotide mix; for the .sup.35 S protocol was 0.5 ul .sup.35
S-dATP (12.5 uCi/ul) also added. In both cases the reaction mixtures, at a
total volume of 10 ul, were incubated 20 min at 37.degree. C.
For the .sup.35 S protocol the following nucleotide mixes were used.
Amix: 62.5 uM dCTP, dGTP, dTTP; 25 uM ddATP.
Cmix: 83 uM dGTP, dTTP; 4 uM dCTP; 50 uM ddCTP.
Gmix: 83 uM dCTP, dTTP; 4 uM dGTP; 150 uM ddGTP.
Tmix: 83 uM dCTP, dGTP: 4 uM dTTP; 125 uM ddTTP.
For the .sup.32 p-labelled primer the following nucleotide mixes were used.
Amix: 83 uM dCTP, dGTP, dTTP; 4 uM dATP; 50 uM ddATP.
Cmix: 83 uM dATP, dGTP, dTTP; 4 uM dCTP; 50 uM ddCTP.
Gmix: 83 uM dATP, dCTP, dTTP; 4 uM dGTP; 50 uM ddGTP.
Tmix: 83 uM dATP, dCTP, dGTP; 4 uM dTTP; 50 uM ddTTP.
After completed reactions the supernatant was removed and the gel was
extensively washed with water. The newly synthesized oligonucleotides were
eluted using 10 ul 0.15M NaOH and the eluant was subsequently neutralized
with 1.25M HAc. The samples were ethanol precipitated and redissolved in 5
ul TE. A fraction of 2 ul was mixed with 2 ul formamide/dye mix and heated
for 3 min in boiling water and loaded on gel a 6% polyacrylamide
sequencing gel. The avidin agarose gel, with immobilized template DNA, was
regenerated by extensive washing with 0.15M NaOH and water.
Solid-Phase Sequencing Using Labelled Deoxy-Nucleotide
The sequencing reactions were performed using a protocol involving
labelling of the specific fragments with .sup.35 S during extention. A
nucleotide mix was used containing, in addition to the standard
nucleotides, the .sup.35 S-labelled dATP and one of the dideoxy
nucleotides. In this and the following experiments, a plasmid was used,
consisting of pRIT27 containing an insert derived from the multi-linker
region of M13 mp 18.
Approximately 1 ug of the plasmid was used for the immobilization to 1 ul
avidin agarose and the subsequent strand specific elution was performed as
described above. A RIT primer (Olson et al, 1986), complimentary to a
region immediately downstream from the multi-linker region, was used to
initiate the extensions. Equimolar amounts of immobilized single stranded
DNA and RIT primer were mixed, incubated for 1 hour at 60.degree. C. and
allowed to cool to room temperature. The supernatant was removed and the
primer extension was started with the appropriated nucleotide mix in a
total volume of 10 ul, followed by a chase reaction (Sanger et al (1977))
to extend fragments not terminated with a dideoxy nucleotide.
After completion of the reactions, the supernatant was removed and the gel
was extensively washed. The newly synthesized oligonucleotides were then
eluted using 0.15M NaOH and the eluant was neutralized with HAc. The
affinity gel containing the single stranded template was thereafter used
for another round of sequencing reactions, involving primer annealing
followed by extension using a new dideoxy nucleotide mix.
The protocol was followed for all four dideoxy nucleotides and the eluted
samples were ehtanol precipitated and re-dissolved in formamide/dye mix
prior to loading a sequencing gel. An autoradiogram of DNA fragments
separated by electrophoresis is presented in FIG. 5A, which also shows the
expected sequence of the plasmid used. Clearly readable sequences are
obtained, which correlates well with the expected ones. The strong band at
the top of the sequence represents run off transcripts at the EcoRI site
at the 5' end of the immobilized template.
Solid-Phase Sequencing With End-Labelled Primer
An alternative strategy was also tested, using a .sup.32 P end-labelled RIT
primer was used to label the extended DNA fragments. A similar protocol
was used, althugh the nucleotide mixes were adjusted appropriately (see
Materials and Methods for details). Different molar ratios of immobilized
template and primer were tested. Equimolar amounts of template and primer
(FIG. 5B) gave a clear and easily readable autoradiogram. Similar results
were obtained for other primer/template ratios (data not shown),
suggesting that the ratio can be varied without critically influencing the
pattern.
The results shown in FIG. 5 demonstrate that the strategy outline in FIG. 1
can be used for solid-phase DNA sequencing using the biotin-avidin system.
EXAMPLE 2
Construction of Plasmid pRIT28
Plasmid pSS1 (see above) was partially digested with PvuII and a synthetic
oligonucleotide linker 5'-GGCCAGGGAGGCCAGATCTGAGCGGCCGCTGCTG-3' (and its
complimentary sequence) was inserted. This fragment contains SfiI, BglII,
EcoB and NotI recognition sequences. The PvuII site used for the insertion
was destroyed simultaneously. The resulting plasmid denoted pRIT28 (FIG.
6), is suitable for solid-phase sequencing using the approach outline in
FIG. 2.
Ligation of Specific Oligonucleotides to Linearized pRIT28
A crucial step in the strategy outline in FIG. 2 is the ligation of
specific oligonucleotides to plasmid DNA linearized with enzymes giving a
3'protruding end (such as SfiI, BstNl etc) and 5' protruding end (such as
Sau3A, TaqI etc). To test the efficiency of ligation, two synthetic
oligonucleotides were synthesized and ligated to pRIT28 cleaved with SfiI
or BamHl. The synthetic SfiI oligonucleotide (5'-TGATCAGGG-3') is in the
3'-end complimentary to one of the strands of pRIT28 after cleavage with
SfiI, while the synthetic BamHl oligonucleotide (5'-GATCAG
CCTTATGTTCATTAG-3') is in the 5'-end complimentary to the 5'-protruding
ends of DNA cleaved with Sau3A, BamHl etc.
To test the efficiency of ligation of each oligonucleotide to pRIT28, the
ligation mixture was analyzed by 1% agarose gel electrophoresis, before
and after ligation. A successful incorporation of oligonucleotide in the
DNA vector will terminate further ligation between plasmids or
circulization of plasmids. This can be observed by comparing the agarose
gel pattern of ligation mixtures.
The synthetic Sau3A oligonucleotide (3'-GATTAC TTTGTATTCCGACTAG-5') was
kinased with T4 polynucleotide kinase in a kinase buffer containing 50 mM
Tris pH 8, 50 mM MgCl.sub.2, 100 mM DTT and 10 mM ATP. 500 ng of pRIT28
was digested with BamHl and mixed with approx. 150 ng of the kinased
oligonucleotide. The mixture was incubated at 14.degree. C. over night
with ligase and a buffer containing 40 mM Tris pH 7.4, 10 mM MgCl.sub.2,
1.0 mM DTT and 0,2 mM ATP. After incubation the mixture was loaded on a 1%
agarose gel.
Also 100 ng of the synthetic SfiI oligonucleotide (5'-TGATCAGGG-3') was
mixed with 500 ng of pRIT28 digested with SfiI. The mixture was incubated
at 14.degree. C. over night with ligase in a buffer containing 40 mM Tris
pH 7.4, 10 mM MgCl.sub.2, 1.0 mM DTT and 0.2 mM ATP. After incubation the
mixture was loaded on a 1% agarose gel.
The results of the agarose gel electrophoresis are presented in Table 1,
which shows the relative amount of vector separating as linearized
plasmid.
TABLE 1
______________________________________
Relative amount of linearized plasmed pRIT28,
as determined by 1% agarose gel electrophoresis,
after cleavage with BamH1 or SfiI, before and after
ligation with Sau3A or SfiI oligonucleotides.
Enzyme Before no Ligation with:
cleavage ligation
oligo SfiI-oligo
Sau3A-oligo
______________________________________
BamH1 100 5 5 95
SfiI 100 5 95 5
______________________________________
The results show that SfiI and Sau3A oligonucleotides efficiently
terminates ligations of pRIT28 cleaved by SfiI and BamHl, respectively.
This demonstrates that the oligonucleotides are efficiently and
specifically incorporated into the site created by SfiI and BamHl,
respectively.
EXAMPLE 3
Solid-Phase DNA Sequencing Using PCR Amplified Templates
Solid-phase sequencing using the concept schematically outlined in FIG. 1
was performed except that the target sequence was amplified by the PCR
technique before immobilization by biotin to avidin agarose. Plasmid
pRIT27 containing a synthetic human proinuslin gene fragment was
transformed in to E.coli strain RR1 M15 and plated on agar medium. A
single colony was picked up with a sterilised Pasteur pipette and
suspended in 10 ul PCR buffer, consisting of 67 mM Tris-HCl, pH 10.00,
16.6 mM (NH.sub.4).sub.2 SO.sub.4, 6.7 mM MgCl.sub.2, 10 mM
.beta.-mercaptoethanol and 170 ug/ml BSA. The sample was heated to
95.degree. C. for 5 min and, after cooling to room temperature,
neutralized by the addition of 1 ul of a 10.times.PCR buffer, pH 7.0.
The PCR was performed with two oligonucleotide primers complimentary to a
region upstream, (biotin-CCATGATTACGAATTTAATAC-3') and downstream
(5'-TTCGATA TCGGTAACCAGCACTCCATGTCATGG-3'), respectively, of the
multi-linker region. The upstream primer was biotinylated in the 5'-end as
described by the manufacturers (Pharmacia, Sweden).
The reaction micture (100 ul) consisted of the above described PCR buffer,
pH 8.8, 1 uM each of the primers, 200 uM each of dATP, dCTP, and dTTP and
the above described 10 ul of lysed sample. Two units of TaqI-polymerase
(Amersham, England) was added and temperature cycle reactions were carried
out using a Techne programmable Dri-Block PHC-1 (Techne, UK). Each cycle
included a heat denaturation step at 92.degree. C. for 1 min, followed by
annealing of primers to the DNA for 2 min at 50.degree. C., and DNA chain
extension with TaqI-polymerase for 1 min at 72.degree. C. The reaction
mixture were covered with a drop of parafin oil. After 20 cycles, the
mixture were added to 20 ul of avidin agarose (as described in example 1).
The supernatant was removed and the immobilized doubled stranded DNA was
converted into single stranded form by incubation at 37.degree. C. with
0.15M NaOH for 15 min. The avidin agarose, with immobilized template DNA
was subsequently washed with 0.15M NaOH and TE-buffer.
Sequencing reactions were performed using a fluorescent end-labelled
sequencing primer (5'-CGTTGTAA AACGGCCAGT-3'), complimentary to a region
immediately downstream from the multi-linker region. 2 pmole of the
sequencing primer were mixed with the avidin agarose immobilized template
DNA in a buffer containing 10 mM Tris-HCl (pH 7.5) 10 mM MgCl.sub.2, 100
ug/ml BSA and 100 mM NaCl to a total volume of 10 ul. The annealing
mixture was heated at 65.degree. C. and allowed to cool to room
temperature. 1 ul DTT/NaCl mixture (0.8M NaCl/0.1M DTT) and 4 units of
T7-polymerase (Pharmacia, Sweden) were added and the volume was adjusted
to 15 ul. Then 3.5 ul of aliquots of the mixture were mixed with 2.5 ul of
respective nucleotide mixture and incubated 10 min at 37.degree. C. The
following nucleotide mixtures were used; 80 uM each dATP, dCTP, dGTP,
dTTP, 6.3 uM of respective ddNTP, 50 mM NaCl and 40 mM Tris-HCl pH 7.5.
When the extension reactions were complete the supernatants of each
reactions were removed and the avidin agarose was washed with water. The
newly synthesized oligonucleotides were eluted using 3 ul of a
formamide/sequencing dye mixture consisting of deionized formamide
containing 10 mM EDTA, pH 7.5. After 15 min incubation at 37.degree. C.
the supernatant was removed and diluted with 3 ul water. Approx 2 ul were
loaded into an automated sequencing apparatus set-up to detect fluroescent
bands during electrophoresis (Ansorge et al). A sequencing run with a 20
cm separating length, and 7% polyacrylamide gel, gave clear results. This
example illustrates that the solid-phase sequencing can be used for
sequencing of PCR amplified DNA using T7 DNA polymerase and a fluorescent
primer.
EXAMPLE 4
Solid Phase Sequencing of Genomic DNA Using PCR Amplified Templates on
Surface Modefied Microtiter Plates
Staphylococcus aureus SA113 was grown as single colonies on TBAB-plates. A
single colony was picked with a sterilized Pasteur pipette and suspended
in 10 ul PCR buffer, consisting of 67 mM Tris-HCl, pH 10.0, 16.6 mM
(NH.sub.4)SO.sub.4, 6.7 mM MgCl.sub.2, 10 mM .beta.-mercaptoethanol and
170 ul/ml BSA. The sample was lysed by heating at 95.degree. C. for 5 min
and, after cooling to room temperature, neutralized by the addition of 1
ul of a 10.times.PCR buffer, pH 7.0.
The PCR was performed with two oligonucleotide primers complimentary to the
Staphylococcal protein A gene. One primer was biotinylated at the 5'-end
(biotin-AATAGCGTGATTTTGCGGT-3'), the second primer
(GACCACCGCATT-GTGGACGTGACCGGCAGCAAAATG-5'), contains, at the 5'-end, a
specif handle sequence not complimentary to the DNA template; this handle
sequence creates a primer annealing seuence.
The reaction mixture (100 ul) consisted of the above described PCR buffer,
pH 8.8 1 uM each of the primers, 200 uM each of dATP, dCTP, dGTP and dTTP
and the above described 10 ul of lysed sample. Two units of
TaqI-polymerase (Amersham, England) were added and temperature cycle
reactions were carried out using a Techne programmable Dri-Block PHC-1
(Techne, UK). Each cycle included a head denaturation step at 92.degree.
C. for 1 min, followed by annealing of primers to the DNA for 2 min at
50.degree. C. and DNA chain extension with TaqI-polymerase for 1 min at
72.degree. C. The reaction mixture was overlaid with a drop of paraffin
oil.
Polystyrene microtiter plates (Costar, USA) were surface grafted with 0.2M
glycidyl methacrylate and 2M benzophenone, in acetone by UV irradiating
for 2 minutes. (K. Almer et.al, Polymer Chamistry 26, 1988, 2099-21110).
25 ug Streptavidin (Amersham, UK) in a TE buffer, at a total volume of 10
ul was applied to each well and the microtiter plate was incubated over
night at 42.degree. C. After removing the supernatant, the wells were
incubated with BSA, 100 ug/ml in TE-buffer, overnight at a total volume of
10 ul and 42.degree. C. The supernatant was removed and the wells
subsequently washed with 1.times.TE.
After 20 temperature reaction cycles, 10 ul of the mixture were added to
each of 4 microtiter wells, prevously treated as above described. After 15
minutes the supernatant was removed and the wells washed with H.sub.2 O.
The immobilized double stranded DNA was converted into single stranded
form by incubation at 37.degree. C. with 15 ul 0.15M NaOH. The wells, with
immobilized template DNA were subsequently washed with 0.15M NaOH and
TE-buffer.
Sequencing reactions were performed using a 32P end-labelled sequencing
primer (5'-GTAAAACGGCCAGT-3'), complimentary to a region immediately
downstream from the multi-linker region. 2 pmole of the sequencing primer
were mixed with the avidin agarose with immobilized template DNA in a
buffer containing 10 mM Tris-HCl (pH 7.5) 10 mM MgCl.sub.2, 200 ug ml BSA
and 100 mM NaCl at a total volume of 10 ul. The annealing mixture was
heated to 65.degree. C. and allowed to cool to room temperature.
The supernatant was removed and 4.2 ul of nucleotide mixture (see below)
were added together with 2 ul DTT/NaCl mixture (0.03M DTT/0.25 mM NaCl)
and 1 unit of T7-polymerase (Pharmacia, Sweden), in a buffer consisting of
10 mM Tris-HCl (pH 7.5), 10 mM MgCl.sub.2, 100 ug/ml BSA and 100 mM NaCl.
The volume was adjusted to 10 ul and the microtiter plate were incubated
10 minutes at 37.degree. C.
The following nucleotide mixture was used; 80 uM each of dATP, dCTP, dGTP,
dTTP and 6.3 uM of respectively ddNTP and 50 mM NaCl and 40 mM Tris-HCl pH
7.5. After completed extension reactions the supernatant was removed and
the microtiter wells were washed with H.sub.20. The newly synthesized
oligonucleotide were eluted using 3 ul of a formamide/sequencing dye
mixture consisting of deionized formamide containing 10 mM EDTA, pH 7.5
0.3% (w/v) xylan cyanol FF and 0.3% (w/v) Bromphenol Blue. After 15 min
incubation at 37.degree. C. the supernatant was removed and diluted with 3
ul H.sub.2 O.
Approx. 2 ul were loaded on a seuencing gel, and the resulting
autoradiogram showed a clear sequence of the Staphylococcal gene fragment.
This example illustrates that the solid phase sequencing approach can be
used for direct sequencing of genomic DNA using PCR technology. It also
illustrated that plastic microtiter wells with covalently bound
streptavidin can be used as a solid support.
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