EP0635065A1

EP0635065A1 - Method of sequencing

Info

Publication number: EP0635065A1
Application number: EP93907982A
Authority: EP
Inventors: Erik Hornes
Original assignee: Dynal AS
Current assignee: Life Technologies AS
Priority date: 1992-04-03
Filing date: 1993-04-02
Publication date: 1995-01-25
Also published as: AU3898293A; CA2117603A1; AU662626B2; GB9207598D0; JPH07505291A; WO1993020232A1

Abstract

The present invention provides a method of sequencing two or more regions of target DNA wherein each of said regions is annealed to a different sequencing primer followed by DNA synthesis using a sequencing polymerase and sequencing nucleotides with subsequent size grading of the DNA so synthesised, each of said sequencing primers being provided with separate distinguishing and/or immobilisation means except that one of said sequencing primers may lack such means, whereby the populations of DNA synthesised from each DNA region may be distinguished and/or separated and a kit for carrying out such a method.

Description

Method of sequencing

This invention relates to a method of simultaneous sequencing of two or more regions of DNA.

There is an increasing demand for reliable sequencing of DNA, particularly in relation to the human genome project and the identification of DNA associated with pathological conditions. It is particularly desirable that such sequencing methods lend themselves to automation.

Recently, many technical improvements have been reported, although the major contributions concern the data evaluation, i.e. computer software. A filter method to prepare 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 ( ada 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 fluorescence 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 being 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) .

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, reproducible reactions and easy automation.

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, site-directed manipulations of cDNA synthesis. 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).

WO 89/09282 describes a method of sequencing one strand of double stranded DNA whereby the latter is immobilised via one terminus of one of the two strands and subjected -to strand separation prior to sequencing the immobilised strand. In order to improve the reliability of the sequence information so obtained, it is possible to sequence the non-immobilised strand in the solution from which the immobilised strand has been removed. However, such a solution is normally at a high pH, due to the conventional use of sodium hydroxide in the strand separation stage, and has to be neutralised. Additionally, it is difficult to avoid diluting the solution containing the non-separated strand to a concentration optimal for sequencing.

It has been proposed in our copending application No. 9122060.8 to carry out sequencing of double stranded DNA by effecting preliminary PCR amplification of target DNA using primers provided with different means for immobilisation, so that the two strands of the amplified DNA can be separated and sequenced independently.

In diagnosis, it is often desirable to sequence more than one target region of DNA eg. from an invading organism or, as indicated above, to sequence both strands of double stranded DNA to obtain improved reliability. In general, particularly in automated systems, it would be advantageous to be able to sequence simultaneously two or more regions of DNA in order to save on operating time and effort.

The present invention is based on the concept of carrying out sequencing of two or more regions of target DNA simultaneously by providing each of the sequencing primers with different means for attachment to immobilising supports and/or labels.

According to the present invention there is provided a method of sequencing two or more regions of target DNA wherein each of said regions is annealed to a different sequencing primer followed by DNA synthesis using a sequencing polymerase and sequencing nucleotides with subsequent size grading of the DNA so synthesised, each of said sequencing primers being provided with separate distinguishing and/or immobilisation means except that one of said sequencing primers may lack such means, whereby the populations of DNA synthesised from each DNA region may be distinguished and/or separated.

The method of the invention may be used to sequence separate regions of DNA on the same strand of DNA or even on separate strands of target DNA.

The method of the invention is particularly suitable for sequencing both strands of target double stranded DNA and according to a further feature of the invention there is provided a method of sequencing both strands of double stranded target DNA wherein said DNA is separated in solution into its two single strands and each strand is annealed to a different sequencing primer followed by DNA synthesis using a sequencing polymerase and sequencing nucleotides with subsequent size grading of the DNA so synthesised, one of said primers being provided with distinguishing or immobilisation means and the other primer lacking such means, or being provided with different distinguishing or immobilisation means, whereby the populations of DNA synthesised from each DNA strand may be distinguished and/or separated.

Where both strands of target DNA are to be sequenced, the choice of polymerase to be used may be related to the method of strand separation applied to the target double stranded DNA. A thermostabile polymerase permits the strands to be separated thermally, eg. at 95°C, and Taq polymerase is useful in such a system. However, thermolabile polymerases such as sequenase, T7 or Klenow cannot be used in this way and a preliminary non-thermal strand splitting step is required. For this purpose, chemical or physico- chemical strand separation is needed, for example ethanol precipitation. Sequenase may be used optimally at about 37°C after a primer annealing step at" about 55°C. Any of such polymerases may be used where strand separation is not required.

Distinguishing means present in the primer and thus incorporated into the DNA which is synthesised may for example, be a fluorescent dye such as , a radioisotope such as ³²P or means for attachment of such labels. In general, where the DNA populations are to be distinguished on the asis of labels (rather than immobilisation) , these will be different substances which may be separately visualised or detected. However, it is also possible to use different quantities of the same label, the distinction being on the basis of the strength of the signal. Thus, for example, if one primer carries a single molecule of label while the other carries two such molecules, it is possible to distinguish the signals both separately (either one or two signal units) or in combination (three signal units) .

In general, sequencing will be by the Sanger method, whereby dideoxy or other 3 '-blocked bases are used together with the four deoxybases needed for synthesis, the dideoxybases causing chain stopping when incorporated. On the basis of competition between the dideoxy bases and the deoxybases, it is found that a complete range of dideoxy-stopped DNA chains is synthesised. Size grading on an appropriate gel is capable of placing the DNA chains in order of molecular weight and knowledge of the terminal dideoxy base in each chain enables the position of each base to be determined.

According to the original Sanger method, the sequencing reaction may be carried out on four aliquots, each aliquot using a single different dideoxybase. The DNA products of the four reactions may be size graded in four separate lanes and the information as to the positions of the separate dideoxy bases may then be integrated to show the complete sequence of the target DNA. In this case, it is possible to label either the sequencing primer, the dideoxy base or even the deoxybases, since all that is required is visualisation of the bands of DNA in each lane of the sizing gel.

More recently, systems have been introduced in which either the primers in each of the four aliquots are separated labelled, normally with four different fluorescent dyes, in which case the dideoxybases are incorporated in four separate reactions and then mixed before loading onto the gel or, more elegantly, the dideoxybases are separately labelled so that they can be incorporated simultaneously in a single reaction. In both cases, size grading can be carried out in a single lane to place the DNA chains in molecular weight order and the different dideoxybases may be directly visualised in their correct positions by the different fluorescent colours associated with each dideoxybase. The initial target DNA may be present in a very small quantity, in which case it may be desirable to amplify it by a preliminary PCR step. The PCR primers may carry DNA extensions which do not hybridise with the - target DNA but hybridise with standard sequencing primers. These will be incorporated into the amplified DNA and thus provide a site for such standard sequencing primers, thereby enabling a standard sequencing system to be used. It will be appreciated that in this case, the amplified DNA carrying such a standard site may be regarded as the target DNA to be sequenced according to the invention.

Immobilisation of the synthesised DNA may be effected in any conventional manner, either batchwise with a suitably activated solid phase slurried in an appropriate medium or on a column of the activated solid phase. Any appropriate solid phase material may be used, eg. Sepharose beads (Pharmacia, Sweden) filters, capillaries, plastic dipsticks or microtitre wells. Magnetic beads, for example the superparamagnetic, monodisperse beads sold by Dynal, Oslo, Norway, are particularly suitable.

Immobilisation means which may be used include biotin (to be used with avidin or streptavidin) , various haptens such as digoxigenin, DNP, NIP or BRDU (to be used with anti-hapten antibodies) and DNA specific to DNA-binding proteins such as Lac operator (to be used with Lac repressor protein) .

A number of embodiments of the invention are described below in greater detail.

A. Where the sequencing primers carry (different) immobilising means, the products of DNA synthesis may be separated into the respective populations from the respective regions of target DNA. Thus, for example, one primer may carry biotin, thus incorporating biotin into one of the two DNA populations and enabling this to be removed from solution by reaction with a solid phase carrying avidin or streptavidin. If another primer carries a different hapten, for example digoxigenin or NPI, this will permit immobilisation using anti-hapten linked to a solid phase. One of the primers may lack attached distinguishing or immobilising means and can thus be identified on this basis.

Before size grading, the immobilised DNA must be liberated into solution. Biotin/avidin or biotin/ streptavidin may be liberated by treatment with formamide. Hapten/antihapten linkages can normally be cleaved under mild conditions by heating or reaction with excess hapten or with an analogue of the hapten which binds more strongly to the antibody.

While immobilisation of the separate DNA populations permits washing of these to be effected prior to size grading, it is, in fact, possible to leave one of the DNA populations free in solution, the residual solution containing this DNA population simply being loaded directly onto the sizing gel. This, of course, avoids the need to remove the latter DNA from a solid phase.

In this way, separate sequencing lanes are produced for each strand and no other distinction is required between the separated DNA populations.

Where four lane sequencing is employed, using a separate DNA synthesis step for each base as described above, it is possible to use the primer to provide the signal which enables the DNA in the gel to be visualised. Although, in the absence of such a signal, the DNA or the gel may be stained subsequently. The primer can thus carry a fluorescent dye, a radioisotope or any label suitable for such visualisation. It is thus possible to attach to a 5'-amino group of the primer both a haptenylated grouping to aid immobilisation and a fluorescent dye to aid visualisation. - 3 -

B. Separation of the DNA populations can be avoided where the primers carry different labels. In this case, the total DNA as synthesised may be loaded onto the sizing gel. It may, however, be convenient for the primers to carry means for attachment to a solid phase, eg. biotin, in order to permit washing of the synthesised DNA prior to size grading.

Where four lane sequencing is used, each lane will show the positions of single base which may be identified as belonging to one or more of the DNA populations by the attached label deriving from the primer, the four lanes thus providing in combination complete information as to the sequences of the separate strands.

C. A combination of sequencing and PCR has recently been proposed (cycling sequencing) whereby the PCR reaction is carried out in the presence of one or more dideoxybases so that an amplified population of dideoxy- stopped DNA chains is produced. This aids sequencing of very small quantities of target DNA. In accordance with the present invention, cycling sequencing can be carried out on both strands of double stranded DNA using sequencing primers as defined above as the PCR primers. As before, different populations of DNA chains will be produced which may be separated and/or distinguished as described previously. In general, it is preferred to use a thermostable polymerase such as Taq I and to carry out a conventional thermal PCR cycle, for example using temperatures as high as 95° for strand separation.

According to a further feature of the invention we provide a kit for carrying out the above sequencing method comprising a sequencing primer provided with distinguishing and/or immobilisation means, a second sequencing primer lacking such means or being provided with different distinguishing or immobilisation means, and optionally at least one of the following: one or more further sequencing primers provided with immobilisation or distinguishing means different from those of said first and second primers; a polymerase; deoxynucleotide triphosphates; dideoxynucleoside triphosphates (optionally labelled) ; sequencing buffer.

The invention will now be described by means of the following non-limiting Example.

EXAMPLE 1 Materials

a) Plasmid pGEM^® 3Zf(+) (Pro ega)

Plasmid pGA7.3 (G. Evensen et al., Journal of Biological Chemistry 266: 6048-6052, 1991) comprising an abrin A gene inserted in vector pGEM^® 7Zf(+) (Promega)

b) Solid supports

- Dynabeads M280-Streptavidin (Dynal)

- Dynabeads coated with anti-Di-Nitro-Phenol (DNP)

c) Anti-DNP (Sigma)

d) Primers used in the sequencing reaction,

5^»DNP-CAG-GAA-ACA-GCT-ATG-AC-3 '

= 5'DNP-rev. seq. primer

5•Biotin GTA-AAA-CGA-CGG-CCA-GT-3 ^•

= 5'Biotin univ. seq. primer e) Buffers

2 x B & W = lOmM Tris HC1 (pH 7.5) lmM EDTA 2.0M NaCl

20 X PBS

adjust pH with NaOH to 7.4 1 x stock is working solution

f) Taq Dye Deoxy™ Terminator Cycle Sequencing Kit Part No. 901497 ABI

Methods

1. Preparation of Dynabeads M-280 Streptavidin

20μl (200μg) of the Dynabeads were washed once in 20μl of 2 x B & W buffer using a Magnet (Dynal MPC- E) and an Eppendorf tube. After washing, the beads were resuspended in 40μl 2 x B & W buffer.

2. Preparation of anti-DNP coated Dynabeads M-280

Anti-DNP (Sigma) and Tosylactivated Dynabeads M-280 (Dynal) were used as described by Dynal in the product package insert.

3. Cycle sequencing protocol

The protocol as suggested by ABI in their part Number 901497 were followed with lμg of plasmid DNA and 3.2 p. ol of primers (in this case of both primers) in a final volume of 20μl.

The cycling reaction was carried out in Perkin- Elmer Thermo cycler 480 with the following cycle:

96°C for 30 seconds 50°C for 15 seconds 60"C for 4 minutes

25 cycles total.

Then rapid thermal ramp to 4°C and hold. The sequencing products were purified using the phenol chloroform extraction protocol suggested in the cycle sequencing kit (ABI) (to remove excess dye terminators). Final volume 40μl.

After purification the sequencing product were immobilized onto Dynabeads M-280.

4. Immobilisation of the sequencing product

The 40μl phenol extracted sequencing product was heated to 90°C for 1 minute and rapid-cooled; added to 40μl of prewashed Dynabeads M-280 streptavidin and incubated at ambient temperature for 15-30 minutes keeping the beads suspended.

After the supernatant_was removed using the MPC-E (Dynal) and saved, 8μl of 0.1M NaOH was added and incubated at ambient temperature for 10 minutes.

5. Separating possible double stranded immobilized product

Using the MPC-E the supernatant containing the eluted material was transferred to a new Eppendorf tube and a new treatment of 8μl O.IM NaOH was performed.

The Dynabeads with the immobilized biotinylated strand were washed once with 50μl O.IM NaOH, once with 40μl B & W and once with 50μl TE buffer. The beads were resuspended in "loading" buffer containing 95% formamide heated to 95°C for minutes and the supernatant was saved for the sequencing gel.

6. Immobilizing the DNP labelled sequencing products

The saved supernatant from step 4 plus 2 x 8μl from step 5 were mixed and neutralised using 16μl O.IM HC1 and 2μl of 1M tris-HCl pH 7.4 the volume was adjusted to 400μl using PBS pH 7.4.

This suspension was heated to 95°C for 1 minute and rapid-cooled on ice.

500μg Dynabeads anti-DNP were washed once in PBS and resuspended in 50μl PBS and added to the neutralized DNP labelled solution. The mixture was incubated at ambient temperature for 30 minutes with occasional mixing, after washing once with lOOμl PBS and 50μl 1 x TE, loading buffer was added, heated to 95"C for 1 minute and the supernatant was ready for gel loading.

7. Gel separation

An ABI 373A DNA sequencer was used. EXAMPLE 2

The plasmid used was as described in Example 1.

1. Template preparation

Target Template: 20ng pGA7.3

Primers: 5'-AAAGGGGGATGTGCTGCAAGGCG-3 '

5 '-GCTTCCGGCTCGTATGTTGTGTG-3 ^•

Polymerase: l units Ampli Taq, Perkin Elmer Cetus

10X buffer with dNTP: 500 M KCl, 100 mM Tris pH 8.3

15 mM MgCl₂, 0.01% gelatin, 2 mM dNTP (ultrapure, Pharmacia) .

Cycling conditions:

The cycling reaction was carried out using a Perkin Elmer 9600 Gene Amp PCR system, with the following cycle:

95"C for 5 seconds 65°C for 1 second 72"C for 1 minute

25 cycles total

Then rapid thermal ramp to 4°C and hold.

Purification of PCR product to remove excess primers:

A Centricon ™100 microconcentrator, Amicon^® was used. The protocol from Applied Biosystems (ABI) was followed:

PCR solution and water up to 2ml was added to the column and spun at 3000g for 10 minutes. The column was turned and spun at 27Og for 2 minutes. 2. Sequencing reactions

The protocol was based on the Taq Dye Deoxy™ Terminator Cycle Sequencing Kit, ABI.

Sequencing mix:

for 10 reactions:

40 μl 5 x TACS buffer (400 mM Tris-HCl, 10 M MgCl₂

100 mM (NH₄)₂S0₄, pH 9.0 10 μl dNTP (750 μM dITP, 1.50 μM dATP, 150 μM dTTF

150 μM dCTP) 10 μl A Dye Deoxy™ Terminator 10 μl C Dye Deoxy™ Terminator 10 μl G Dye Deoxy™ Terminator 10 μl T Dye Deoxy™ Terminator 5 μl Ampli Taq¹⁸ DNA polymerase, 8 V/μl

(1) DNP-labelled universal sequencing primer

5' DNP-DNP-DNP-GTA AAA CGA CGG CCA GT-3 ^■

(2) Biotin-labelled reversed sequencing primer

5' Biotin-CAG GAA ACA GCT ATG AC-3 ' .

Two separate reactions using the above primers were run under the following conditions:

Iμl purified pGA7.3 PCR product 2μl DNP primers (1.6 pmol/μl) lμl Biotin primer (4.8 pmol/μl) 9.5 μl Sequencing mix 6.5 μl dist. water.

The cycling reaction was carried out using the Perkin Elmer 9600 system with the following cycle 96°C for 15 seconds 50°C for 1 second 60°C for 4 minutes

25 cycles total.

Then rapid thermal ramp to 4"C and hold.

3. Purification of sequencing product

80 μl of H₂0 was added to each reaction before phenol extraction. Prior to use, the phenol was buffered with 1M Tris pH 8.0 and lxTE. Each sample was extracted 2x with 100 μl phenol: chloroform : water (68 : 14 : 18) . The water phase was precipitated with 10 μl 3M sodium acetate and 300 μl ethanol (EtOH) , spun at 17000g for 30 minutes at 4°C. The pellet was washed with 250 μl 70% EtOH, spun for 5 minutes as above and dried for 3 minutes using Speed Vac.

4. Separation of sequencing products

Each pellet was dissolved in 40 μl PBS buffer (50 x PBS buffer (3.0M NaCl, 0.2M NaH₂P0₄ pH 6.4)) and the two were mixed together.

The solid support:

(a) Dynabeads M280 Streptavidin (available from Dynal AS)

(b) Dynabeads M280 anti DNP, prepared by incubating Dynabeads M280 RAM G2a with anti DNP, mouse IgG, 2A (Oswell DNA Service Edinburgh) as recommended by the supplier (DYNAL) .

The following separation strategies were tested: 12. A: 40 μl sequencing product was heated to 100"C for 10 minutes, and placed directly on ice for 2 minutes.

2mg Dynabeads M280 anti DNP previously washed in PBS and suspended in 60 μl PBS were added and put on a roller for 30 minutes at room temperature (RT) . The supernatant was transferred to another tube.

5 μl formamide : EDTA were added and boiled for 10 minutes. The supernatant was loaded on the sequencing gel. This fraction contains the DNP labelled sequencing products.

The first collected supernant was added to 100 μl 0.2M NaOH, 2M NaCl and held at RT for 5 minutes. 400 μg Dynabeads M280 Streptavidin were added and placed on a roller for 30 minutes at RT. The supernatant was removed and to the Dynabeads were added together 5 μl Formamide : EDTA, heated at 100°C for 10 minutes. The supernatant was loaded on the sequencing gel.

This fraction contains the Biotin-labelled sequencing products.

A typical result is shown in Tables 1 and 2 (12a DNP and 12a SA respectively) .

12. B: To 40 μl sequencing product was added 4 μl IM NaOH and placed at RT for 5 minutes. 400 μg Dynabeads M280 Streptavidin in 56 μl PBS were added and finally another 7 μl IM NaOH was added and placed on a roller for 30 minutes at RT. The supernatant was transferred to another tube and 5 μl Formamide: EDTA was added to the Dynabeads and heated to 100°C for 10 minutes. The Supernatant was loaded on the sequencing gel. This contains the Biotin-labelled sequencing product.

To the first supernatant was added 30 μl 0.33M HC1, and the pH adjusted to 8-9. 100 μl PBS was added and boiled for 10 minutes then placed on ice for 2 minutes. 2mg Dynabeads M280 anti DNP were added and placed on a roller for 30 minutes at RT. The supernatant was removed and 5μl formamide : EDTA was added and boiled for 10 minutes. The supernatant was loaded on the sequencing gel.

This contains the DNA-labelled sequencing product. All reactions were run on an ABI 373A DNA sequencer.

A typical result is shown in Table 1 and 2 (12b DNA and 12b SA, respectively) .

The separation strategies for the complex sequencing solution are the same regardless of sequencing strategy.

Table 1

Table 2

Claims

1. A method of sequencing two or more regions of target DNA wherein each of said regions is annealed to a different sequencing primer followed by DNA synthesis using a sequencing polymerase and sequencing nucleotides with subsequent size grading of the DNA so synthesised, each of said sequencing primers being provided with separate distinguishing and/or immobilisation means except that one of said sequencing primers may lack such means, whereby the populations of DNA synthesised from each DNA region may be distinguished and/or separated.

2. A method as claimed in claim 1 for sequencing both strands of double stranded target DNA, in which said DNA is separated in solution into its two single strands and each strand is annealed to a different sequencing primer followed by DNA synthesis using a sequencing polymerase and sequencing nucleotides with subsequent size grading of the DNA so synthesised, one of said primers being provided with distinguishing or immobilisation means and the other primer lacking such means, or being provided with different distinguishing or immobilisation means, whereby the populations of DNA synthesised from each DNA strand may be distinguished and/or separated.

3. A method as claimed in claim 1 in which said regions occur separately on the same DNA strand.

4. A method as claimed in claim 2 in which the strand separation is effected by heating and the sequencing polymerase is a thermostable polymerase.

5. A method as claimed in any of claims 1 to 4 in which said means for distinguishing comprise a fluorescent dye or a radionucleide.

6. A method as claimed in any of claims 1 to 5 in which said means for immobilisation is biotin, a hapten or a protein-binding DNA sequence.

7. A method as claimed in any of claims 1 to 6 in which both dideoxynucleotides and deoxynucleotides are present to produce said populations of DNA by chain stopping at different chain lengths.

8. A method as claimed in claim 7 in which the dideoxybases are labelled with different fluorescent dyes and the sequencing primers are provided with different immobilising means whereby DNA populations containing different primers may be separated and size- graded.

9. A method as claimed in any of the preceding claims in which one or more of said primers carries both means for immobilisation and means providing a visual signal.

10. A method as claimed in any of the preceding claims in which the initial target DNA is subjected to amplification.

11. A method as claimed in claim 10 in which amplification and sequencing are effected simultaneously by carrying out PCR amplification using primers as defined in claim 1 in the presence of dideoxy nucleotides and deoxynucleotides.

12. A kit for carrying out the method as claimed in claim 1 comprising a first sequencing primer provided with distinguishing and/or immobilisation means, a second sequencing primer lacking such means or being provided with different distinguishing or immobilisation means, and optionally at least one of the following: one or more further sequencing primers provided with immobilisation or distinguishing means different from those of said first and second primers; a polymerase; deoxynucleotide triphosphates; dideoxynucleoside triphosphates (optionally labelled) ; sequencing buffer.