Process for sequencing nucleic acids

For the sequencing of nucleic acids by enzymatic extension of an oligonucleotide primer in the presence of a polymerase, the four deoxyribonucleoside triphosphates, one of the four dideoxynucleoside triphosphates and the nucleic acid to be sequenced as template in each of four different preparations, labelling of the nucleic acid fragments which form and which are dependent on the dideoxynucleoside triphosphate used, separation by gel electrophoresis of the preparations containing the fragments and detection of the sequence via the label, at least two of the dideoxynucleoside triphosphates are used in different amounts together in one preparation and the fragments are differentiated by the intensity of the labelling signal of the bands in the gel or in another separation procedure.

DESCRIPTION 
The invention involves an improvement on a process for sequencing nucleic 
acids. The process which is improved is one in which an oligonucleotide 
primer is extended in the presence of (i) a polymerase, (ii) all four 
deoxyribonucleoside triphosphaes, (iii) a single dideoxynucleoside 
triphosphate, and (iv), the nucleic acid to be sequenced, which acts as a 
template. In this known process, four different preparations, each of 
which uses a different dideoxyribonucleoside are required. Nucleic acid 
fragments form, which are then labelled. The fragments which form depend 
upon which of the dideoxyribonucleosides is used. The fragments thus 
formed are separated via gel electrophoresis, and the fragment is then 
determined via its incorporated label. 
In order to sequence DNA, two methods are generally known. The first is the 
chemical degradation procedure according to Maxam and Gilbert (A. M. Maxam 
and W. Gilbert, Proc. Natl. Acad. Sci. USA 74 (1977), 560 and A. M. Maxam 
and W. Gilbert, Methods in Enzymol. vol. 65, (1980) p. 499). The second is 
and the enzymatic dideoxy chain termination method (F. Sanger et al., 
Proc. Natl. Acad. Sci. USA 74 (1977), 5463-5467). Using the method 
according to Sanger one starts with a DNA template, and produces many 
labelled DNA molecules of differing length by enzymatic extension of a 
synthetic primer, using DNA polymerase and a mixture of deoxy- and 
dideoxynucleoside triphosphates. To do this, a mixture of a certain 
deoxynucleoside triphosphate and a corresponding dideoxynucleoside 
triphosphate together with the three other deoxynucleoside triphosphates 
is used in each of four preparations. Each preparation contains a 
different dideoxynucleoside. In this way statistical incorporation of the 
dideoxynucleotides into the growing DNA chain is achieved, because after a 
dideoxynucleotide is incorporated into a DNA chain, it cannot grow any 
longer because of the absence of a 3'-OH group. Thus, many DNA fragments 
are formed which from a statistical point of view, contain at least one 
dideoxynucleoside at every possible incorporation site and which end 
there. These four preparations with fragmetns each ending at the positions 
of one base are each separated in one lane on polyacrylamide gels and the 
sequence is determined after autoradiography. 
In the Maxam-Gilbert method end-labelled DNA molecules are modified 
chemically in a base-specific manner, partial strand termination is 
effected and the fragments thus obtained are separated by polyacrylamide 
gel electrophoresis. The sequence is determined after autoradiography. 
Both known methods have advantages and disadvantages. An advantage of the 
Sanger dideoxy method is that the label and the production of 
base-specific fragments can be combined in one step, Also, single-stranded 
as well as double-stranded DNA can be sequenced. Furthermore, the 
sequencing of longer DNA fragments is made possible by so-called 
"shot-gun" experiments in M13-descendants etc. Until about 1986, DNA 
sequencing was carried out using radioactive (.sup.32 P or .sup.35 S) 
labels. After gel electrophoresis and autoradiography the nucleotide 
sequence was determined either manually or semi-automatically. Since then, 
fluorescence labelling has also been used for dideoxy sequencing (L. M. 
Smith et al., Nature, 321 (1986) 674-679; W. Ansorge et al., J. Biochem. 
Biophys. Meth. 13 (1986) 315-323). Using this procedure, the nucleotide 
sequence can be read automatically during the electrophoresis and can be 
directly entered into a computer. 
In contrast the Maxam-Gilbert method is used reluctantly for sequencing 
long DNA fragments because of the following disadvantages: the labelling 
of the DNA and the formation of base-specific fragments have to be carried 
out in two separate steps and, the available labelling techniques are 
complicated and require the presence of suitable restriction sites on the 
nucleic acids to be sequenced. In addition to this, four steps have to be 
carried out for standard labelling methods according to Maxam and Gilbert, 
i.e. the cleavage of DNA with a restriciton endonuclease, the enzymatic 
labelling, cleavage of the labelled DNA fragments with a second 
restriction endonuclease so that the labelled DNA fragments are only 
labelled on one end and isolation of the DNA fragments which are only 
labelled at one end by agarose- orpolyacrylamide gel electrophoresis. 
Further disadvantages of the Maxam-Gilbert method include the fact that 
sequencing of single-stranded DNA is not easy to carry out, and random or 
systematic strategies, as they are carried out in connection with the 
enzymatic dideoxy method, cannot be used for sequencing long DNA 
fragments. In addition, the chemical reactions in solution (as described 
by Maxam and Gilbert) are laborious because of the precipitation steps 
needed. The Maxam and Gilbert process has only been described in 
connection with radioactive labelling. 
As a result of the disadvantages of the Maxam-Gilbert method, sequencing of 
DNA nowadays is usually carried out according to the method of Sanger et 
al. Both methods present a serious disadvantage in that this method 
requires the four different extension reactions to be carried out in 
different preparations and loaded on different lanes of a gel for gel 
electrophoresis. Because of a this only the fragments of relatively few 
sequencing preparations can be loaded on a gel carrying out four different 
preparations for every nucleic acid to be sequenced is exceedingly 
laborious and time-consuming. 
it is therefore the object of the present invention to avoid these 
disadvantages of the sequencing method according to Sanger et al., and to 
make available a sequencing method which can be carried out quickly and 
simply based on the well known method according to Sanger et al. 
The object of the present invention is therefore a process for sequencing 
nucleic acids by enzymatic extension of an oligonucleotide primer in the 
presence of a polymerase, the four deoxyribonucleoside triphosphates, one 
of the four dideoxynucleoside triphosphates and the nucleic acid to be 
sequenced as template in each of four different preparations, labelling of 
the nucleic acid fragments which form and which are dependent on the 
dideoxynucleoside triphosphate used, separation of the preparations 
containing the fragments by gel electrophoresis and detection of the 
sequence via the label, characterized in that at least two of 
dideoxynucleoside triphosphates are used in different amounts in one 
preparation, wherein the fragments are differentiated by the intensity of 
the labelling signal of the bands in the gel or in another separation 
procedure. 
The process according to the present invention allows sequencing to be 
carried out in for example only two preparations or only one each of which 
contains two of the dideoxynucleoside triphosphates and which consequently 
are loaded on only two lanes of a gel. This is made possibly by the use of 
the dideoxynucleoside triphosphates in different amounts so that in the 
separation procedure the bands of the fragments which result from the use 
of the one dideoxynucleoside triphosphate occur more frequently than bands 
of fragments with terminations at the positions of the dideoxynucleoside 
triphosphate present in a smaller amount. Thus in the sequence detection 
the bands can be assigned to the bases of the sequence by means of the 
signal intensity of the label. 
In a preferred embodiment of the invention two dideoxynucleoside 
triphosphates are used together in a preparation in a quantity ratio of at 
least 2:1. 
In a particularly preferred embodiment of the invention all four 
dideoxynucleoside triphosphates are used in one preparation in a ratio of 
at least 4:3:2:1. By use of this particularly preferred procedure the 
sequencing of nucleic acids is possible in only one preparation and after 
loading on only one lane of a sequencing gel. The ratio of 4:3:2:1 is the 
lowest limit and, in order to increase the accuracy of the sequencing, 
ratios with a greater difference between the individual dideoxynucleoside 
triphosphates should be used. These differences in the ratio of the 
dideoxynucleoside triphosphates also depend on the polymerase enzyme used 
because enzymes which yield uniform peak patterns can be used with a lower 
ratio of the deoxynucleoside triphosphates than polymerase which do not 
yield such uniform peaks. 
Even though all labelling methods for the sequencing according to Sanger 
are suitable for the procedure according to the present invention such as 
e.g. radioactive labelling, it is preferable to use a fluorescent dye for 
the label. In a particularly preferred embodiment of the invention a 
primer coupled to a fluorescent dye is used for the label. The fluorescent 
dye is preferably bound to the 5'-phosphate group of the primer via a 
linker (FIG. 1A). 
In another particularly preferred embodiment of the invention a deoxy- or 
dideoxynucleoside triphosphate bound to a fluorescent dye via a linker is 
used for the label. In this connection it is especially preferred to use a 
labelled deoxynucleoside triphosphate since the nucleic acid fragment 
which forms can thus be labelled several times whereas when labelling via 
dideoxynucleoside triphosphates or via a primer with a fluorescent label 
at the 5'-phosphate group only one fluorescent dye group is bound to each 
nucleic acid fragment which forms. The intensity of the signal can 
therefore be increased using this preferred embodiment of the invention. 
When using labelled deoxy- or dideoxynucleoside triphosphates it is 
preferably to use those in which the dye is coupled via a linker to the C5 
position of pyrimidines or to the N7, C8 or C7 position of pmrines. 
Within the scope of the invention straight-chain or branched amino- or 
mercapto-hydrocarbon units with more than two carbon atoms in the 
unbranched chain are preferably used as the linker. Especially preferred 
for this are aminoalkyl, aminoalkenyl or aminoalkynyl groups. 
Within the scope of the invention single- or double-stranded nucleic acids 
can be used as the nucleic acids to be sequenced. According to the present 
invention fluorescein, analogues thereof or rhodamine are preferably used 
as markers. Fluorescein is particularly preferred. 
In a preferred embodiment, the process according to the present invention 
is carried out in such a way that two different dideoxynucleoside 
triphosphates in a ratio of 5:1, 6:1 or 7:1 are used in each of two 
preparations. In this way, the strongest bands in each of the two lanes of 
the gel on which both preparations have been loaded can be identified as 
belonging to the dideoxynucleoside triphosphates that have been used in a 
larger amount and the other bands belong to the bases whose 
dideoxynucleoside triphosphate was used in the smaller amount. 
In another particularly preferred embodiment of the invention all four 
different dideoxynucleoside triphosphates are used in only one preparation 
in a ratio of 16:8:4:1. Using these ratios, differentiation of the 
dideoxynucleoside triphosphates is possible and therefore the base 
sequence can be easily read. 
According to the present invention it is also possible to carry out the 
sequencing with only three of the four differential dideoxynucleoside 
triphosphates. In this connection either all three dideoxynucleoside 
triphosphates are used in one preparation or else two dideoxynulceoside 
triphosphates are used in one preparation and only one dideoxynucleoside 
triphosphate is used in a second preparation. After separation, for 
example in a polyacrylamide gel, bands with different signal intensities 
appear in regular intervals whereby a gap occurs in the band pattern at 
the position of the base in the nucleic acid to be sequenced corresponding 
to the missing dideoxynucleoside triphosphate. 
For the extension of nucleotide sequences according to the present 
invention the Klenow fragment of the DNA polymerase I, modified or 
unmodified T7 DNA polymerase, Taq polymerase or reverse transcriptase are 
preferred. Of these polymerases, unmodified T7 DNA polymerase is 
especially preferable because it results in a particularly uniform peak 
pattern. 
In a scope of the invention, it may be necessary, or preferred, to increase 
or to amplify the amount of double stranded nucleic acid to be sequenced 
and which serves as template. This can be done via use of, e.g., the 
polymerase chain reaction (PCR) as taught in U.S. Pat. Nos. 4,683,195; 
4,683,202 and 4,800,159. In this method, the double stranded nucleic acid 
to be sequenced is combined with two synthetic primers in the presence of 
all four deoxynucleosides triphosphates and a polymerase. A cycle takes 
place, and is repeated many times. The "cycle" involves extending the 
primer, heating to separate strands, adding new polymerase, separating the 
extension products of both strands, and then using one strand for 
sequencing. This methodology allows one to sequence, e.g., DNA, which is 
present in only small quantities in the sample. 
The method of detection according to the present invention corresponds to 
the type of label used in each case. Radioactively labelled fragments are 
thus visualized by autoradiography of the gel. In the preferred embodiment 
the present invention a fluorescent dye is used which is excited 
preferably by a laser after separation of the fragments by gel 
electrophoresis and by this means a particularly strong signal is 
obtained. Using the preferred detection method according to the present 
invention the detection can also be carried out in an apparatus which 
detects the bands simultaneously with the gel electrophoresis and 
immediately transmits them to a computer which automatically prints out 
the sequence of the nucleic acid to be sequenced. 
The process according to the present invention allows many different 
nucleic acids to be rapidly and easily sequenced side by side and to be 
simultaneously loaded on a gel. It is also possible, after the run has 
been completed to reload the single lanes of the gel with a new nucleic 
acid to be sequenced by use of the preferred fluorescent labelling and the 
automatic detection. Also by this means the sequencing of many different 
DNA fragments can be accelerated decisively.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
EXAMPLE 1 
a) Fluorescence labelling of the primer 
5'-(S-triphenylmethyl)-3-mercaptopropylphospho)d[GTAAAA-CGACGGCCAGT] was 
synthesized and purified as described by Ansorge et al. in J. Biochem. 
Biophys. Met. 13 (1986), 315-322. 500 nmol silver nitrate was added to 100 
nmol of this compound in 0.1 mol/l triethylammonium acetate solution (1 
ml, pH 7) and left to stand for 1 hour in an Eppendorf tube. Dithiotreitol 
(700 nmol) in 100 .mu.l water was added to this and the contents of the 
tube were mixed and left to stand for 30 minutes. The insoluble silver 
salt was removed by centrifugation. The supernatant was transferred into a 
new Eppendorf tube and 1 mol/l aqueous sodium bicarbonate solution was 
added in order to yield a pH value of 8.5. A solution of 3 mg (6 .mu.mol) 
5-iodoacetamidofluorescein (obtained from Molecular Probes, Inc., Eugene, 
Oreg., USA) in N,N-dimethylformamide (300 .mu.l) was added to this and 
carefully mixed; then it was kept in the dark for 6 hours. Afterwards 10 
.mu.l 2-mercaptoethanol was added in order to remove excess iodoacetamide 
and then the solution was dialyzed in the dark against water (3.times.2 
l). The solution was concentrated in a vacuum and the primer labelled with 
dye was purified by ion-exchange HPLC on a Partisil-Sax column using a 
concentration gradient of potassium dihydrogenphosphate, pH 6.3 in the 
form of amide/water (6:4 v/v) as eluting agent. This process removes a 
large part of the strongly associated but not covalently bound dye. The 
product was desalted by dialysis and further purified by reverse-phase 
HPLC on a C8-Aquapor RP 300 column using 0.1 mol/l triethylammoniumacetate 
pH 7/acetonitrile as eluting agent. The completely purified 
fluorescein-labelled primer was further desalted by dialysis and kept in 
the dark at -20.degree. C. The yield of dye-labelled primer was 15% based 
on the S-trityl-material. In the same way a primer labelled with rosin was 
obtained with a yield of 27%. 
b) Sequencing reaction 
At first in two preparations 1 .mu.l primer solution, about 1 to 2 .mu.g 
single-stranded M13mp18DNA and sequencing buffer were added together to a 
centrifuge tube to give a total volume of 10 .mu.l to 2 .mu.g M13mp18DNA 
in a volume of 7 .mu.l was used as a control. The tubes were heated for 2 
min to 65.degree. C. then cooled down to room temperature over a period of 
30 min. During the cooling the primer hybridizes to its homologous 
position of the M1--mp18DNA. The hybridization is completed when the 
temperature has fallen to below 35.degree. C. The following solutions were 
prepared for the actual sequencing: 
______________________________________ 
Solution T,C: 
25 .mu.l 
0.5 mmol/l dTTP, 
25 .mu.l 
0.5 mmol/l dCTP, 
500 .mu.l 
0.5 mmol/l dGTP and 
500 .mu.l 
0.5 mmol/l dATP, 
5 .mu.l 10.0 mmol/l ddTTP, 
1 .mu.l 10.0 mmol/l ddCTP and 
500 .mu.l 
TE-buffer 
Solution G,A: 
25 .mu.l 
0.5 mmol/l dGTP, 
25 .mu.l 
0.5 mmol/l dATP, and 
500 .mu.l 
each of 0.5 mmol/l dTTP and dCTP, 
5 .mu.l 10 mmol/l ddACT and 
1 .mu.l 10 mmol/l ddGTP together with 
500 .mu.l 
TE-buffer. 
______________________________________ 
Either 2 .mu.l of solution T,C or 2 .mu.l of solution G,A was added to the 
two samples hybridized with the labelled primer. Afterwards they were 
incubated for 20 min at room temperature, then 2 .mu.l formamide was added 
and they were heated for 2 min to 95.degree. C., cooled on ice and loaded 
on a polyacrylamide gel. FIG. 2 shows a graph of the intensities of the 
fluorescein, measured in a computer, when the fluoroscein had been excited 
by a laser for the region of the DNA sequence shown above. For comparison 
FIG. 3 shows part of the M13mp18DNA in which the sequenced region shown in 
FIG. 2 is underlined. In the upper curve of FIG. 2 the peaks are shown for 
fragments which result on addition of ddATP and ddGTP in a ratio of 5:1 in 
one preparation; in the lower graph the peaks are shown for fragments 
which result on addition of ddTTP and ddCTP in a ratio of 5:1 in one 
preparation.