WO1993020232A1 - Method of sequencing - Google Patents
Method of sequencing Download PDFInfo
- Publication number
- WO1993020232A1 WO1993020232A1 PCT/GB1993/000697 GB9300697W WO9320232A1 WO 1993020232 A1 WO1993020232 A1 WO 1993020232A1 GB 9300697 W GB9300697 W GB 9300697W WO 9320232 A1 WO9320232 A1 WO 9320232A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sequencing
- dna
- primers
- immobilisation
- different
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Definitions
- This invention relates to a method of simultaneous sequencing of two or more regions of DNA.
- 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.
- 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.
- 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.
- 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.
- 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.
- thermostabile polymerase permits the strands to be separated thermally, eg. at 95°C, and Taq polymerase is useful in such a system.
- thermolabile polymerases such as sequenase, T7 or Klenow cannot be used in this way and a preliminary non-thermal strand splitting step is required.
- 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 32 P or means for attachment of such labels.
- a fluorescent dye such as , a radioisotope such as 32 P or means for attachment of such labels.
- these will be different substances which may be separately visualised or detected.
- it is also possible to use different quantities of the same label the distinction being on the basis of the strength of the signal.
- 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) .
- 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.
- dideoxy bases 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.
- 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.
- 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.
- 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.
- 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) .
- 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.
- 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.
- 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.
- 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.
- 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.
- the primers carry different labels.
- 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.
- 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.
- 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.
- cycling sequencing can be carried out on both strands of double stranded DNA using sequencing primers as defined above as the PCR primers.
- sequencing primers as defined above as the PCR primers.
- different populations of DNA chains will be produced which may be separated and/or distinguished as described previously.
- 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.
- 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)
- Anti-DNP Sigma
- Tosylactivated Dynabeads M-280 Dynal were used as described by Dynal in the product package insert.
- the 40 ⁇ l phenol extracted sequencing product was heated to 90°C for 1 minute and rapid-cooled; added to 40 ⁇ l of prewashed Dynabeads M-280 streptavidin and incubated at ambient temperature for 15-30 minutes keeping the beads suspended.
- the Dynabeads with the immobilized biotinylated strand were washed once with 50 ⁇ l O.IM NaOH, once with 40 ⁇ l B & W and once with 50 ⁇ l 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.
- step 4 The saved supernatant from step 4 plus 2 x 8 ⁇ l from step 5 were mixed and neutralised using 16 ⁇ l O.IM HC1 and 2 ⁇ l of 1M tris-HCl pH 7.4 the volume was adjusted to 400 ⁇ l using PBS pH 7.4.
- This suspension was heated to 95°C for 1 minute and rapid-cooled on ice.
- Dynabeads anti-DNP were washed once in PBS and resuspended in 50 ⁇ l 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 lOO ⁇ l PBS and 50 ⁇ l 1 x TE, loading buffer was added, heated to 95"C for 1 minute and the supernatant was ready for gel loading.
- the plasmid used was as described in Example 1.
- the cycling reaction was carried out using a Perkin Elmer 9600 Gene Amp PCR system, with the following cycle:
- the protocol was based on the Taq Dye DeoxyTM Terminator Cycle Sequencing Kit, ABI.
- 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
- PBS buffer 50 x PBS buffer (3.0M NaCl, 0.2M NaH 2 P0 4 pH 6.4)) and the two were mixed together.
- the solid support is a solid support
- 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 first collected supernant was added to 100 ⁇ l 0.2M NaOH, 2M NaCl and held at RT for 5 minutes. 400 ⁇ g 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 ⁇ l 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.
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU38982/93A AU662626B2 (en) | 1992-04-03 | 1993-04-02 | Method of sequencing |
JP5517258A JPH07505291A (en) | 1992-04-03 | 1993-04-02 | Sequencing method |
EP93907982A EP0635065A1 (en) | 1992-04-03 | 1993-04-02 | Method of sequencing |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9207598.5 | 1992-04-03 | ||
GB929207598A GB9207598D0 (en) | 1992-04-03 | 1992-04-03 | Method of sequencing double stranded dna |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1993020232A1 true WO1993020232A1 (en) | 1993-10-14 |
Family
ID=10713606
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1993/000697 WO1993020232A1 (en) | 1992-04-03 | 1993-04-02 | Method of sequencing |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0635065A1 (en) |
JP (1) | JPH07505291A (en) |
AU (1) | AU662626B2 (en) |
CA (1) | CA2117603A1 (en) |
GB (1) | GB9207598D0 (en) |
WO (1) | WO1993020232A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1998031833A1 (en) * | 1997-01-15 | 1998-07-23 | Incyte Pharmaceuticals, Inc. | Nucleic acid sequencing with solid phase capturable terminators |
US5976802A (en) * | 1995-04-27 | 1999-11-02 | Europasches Laboratorium fur Molekularbiologie (EMBL) | Simultaneous sequencing of nucleic acids |
WO2000015842A1 (en) * | 1998-09-15 | 2000-03-23 | Dynal Asa | Method of isolation primer extension products with modular oligonucleotides |
US6291164B1 (en) | 1996-11-22 | 2001-09-18 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
US6482592B1 (en) | 1996-09-26 | 2002-11-19 | Dynal As | Methods and kits for isolating primer extension products using modular oligonucleotides |
US6787349B1 (en) | 1999-11-19 | 2004-09-07 | Hitachi Software Engineering Co., Ltd. | Biochip reader and labeling reagent |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0303459A2 (en) * | 1987-08-11 | 1989-02-15 | The President And Fellows Of Harvard College | Multiplex sequencing |
EP0371437A2 (en) * | 1988-11-29 | 1990-06-06 | Orion-Yhtymà Oy | Method and reagent combination for determining nucleotide sequences |
EP0437774A1 (en) * | 1990-01-17 | 1991-07-24 | Roche Diagnostics GmbH | Process for the preparation of modified nucleic acids |
WO1993008305A1 (en) * | 1991-10-17 | 1993-04-29 | Dynal As | Method of sequencing double stranded dna |
-
1992
- 1992-04-03 GB GB929207598A patent/GB9207598D0/en active Pending
-
1993
- 1993-04-02 EP EP93907982A patent/EP0635065A1/en not_active Ceased
- 1993-04-02 JP JP5517258A patent/JPH07505291A/en active Pending
- 1993-04-02 CA CA002117603A patent/CA2117603A1/en not_active Abandoned
- 1993-04-02 AU AU38982/93A patent/AU662626B2/en not_active Ceased
- 1993-04-02 WO PCT/GB1993/000697 patent/WO1993020232A1/en not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0303459A2 (en) * | 1987-08-11 | 1989-02-15 | The President And Fellows Of Harvard College | Multiplex sequencing |
EP0371437A2 (en) * | 1988-11-29 | 1990-06-06 | Orion-Yhtymà Oy | Method and reagent combination for determining nucleotide sequences |
EP0437774A1 (en) * | 1990-01-17 | 1991-07-24 | Roche Diagnostics GmbH | Process for the preparation of modified nucleic acids |
WO1993008305A1 (en) * | 1991-10-17 | 1993-04-29 | Dynal As | Method of sequencing double stranded dna |
Non-Patent Citations (1)
Title |
---|
NUCLEIC ACIDS RESEARCH. vol. 19, no. 12, 25 June 1991, ARLINGTON, VIRGINIA US pages 3301 - 3305 M. CHEE * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5976802A (en) * | 1995-04-27 | 1999-11-02 | Europasches Laboratorium fur Molekularbiologie (EMBL) | Simultaneous sequencing of nucleic acids |
US6482592B1 (en) | 1996-09-26 | 2002-11-19 | Dynal As | Methods and kits for isolating primer extension products using modular oligonucleotides |
US6291164B1 (en) | 1996-11-22 | 2001-09-18 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
US6764839B2 (en) | 1996-11-22 | 2004-07-20 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
US7344835B2 (en) | 1996-11-22 | 2008-03-18 | Invitrogen Corporation | Methods for preventing inhibition of nucleic acid synthesis by pyrophosphate |
WO1998031833A1 (en) * | 1997-01-15 | 1998-07-23 | Incyte Pharmaceuticals, Inc. | Nucleic acid sequencing with solid phase capturable terminators |
WO2000015842A1 (en) * | 1998-09-15 | 2000-03-23 | Dynal Asa | Method of isolation primer extension products with modular oligonucleotides |
US6787349B1 (en) | 1999-11-19 | 2004-09-07 | Hitachi Software Engineering Co., Ltd. | Biochip reader and labeling reagent |
Also Published As
Publication number | Publication date |
---|---|
JPH07505291A (en) | 1995-06-15 |
CA2117603A1 (en) | 1993-10-14 |
AU662626B2 (en) | 1995-09-07 |
GB9207598D0 (en) | 1992-05-20 |
AU3898293A (en) | 1993-11-08 |
EP0635065A1 (en) | 1995-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6090935A (en) | Isolation of nucleic acid | |
US5476769A (en) | Method for assays of nucleic acid, a reagent combination and a kit therefor | |
EP0371437B1 (en) | Method and reagent combination for determining nucleotide sequences | |
WO1991011533A1 (en) | Method for isolating primer extension products from template-directed dna polymerase reactions | |
JPH04503158A (en) | Detection of nucleic acid sequences or changes therein | |
WO1993008305A1 (en) | Method of sequencing double stranded dna | |
EP1301623A2 (en) | Microarray-based analysis of polynucleotide sequence variations | |
EP0777747A1 (en) | Nucleotide sequencing method | |
JP2000500024A (en) | Nucleotide sequencing | |
EP1162278B1 (en) | Method for quantification of a specific nucleic acid by isometric primer extension | |
AU662626B2 (en) | Method of sequencing | |
EP1275738A1 (en) | Method for random cDNA synthesis and amplification | |
EP1275734A1 (en) | Method for random cDNA synthesis and amplification | |
WO1989009281A1 (en) | Method for amplifying and detecting nucleic acid in a test liquid | |
EP0421469B1 (en) | Method for separating a target oligonucleotide | |
WO1995030025A1 (en) | Detection or assay of target nucleic acids | |
EP1679381A1 (en) | Isometric primers extension method and kit for detection and quantification of specific nucleic acid |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1993907982 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2117603 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 1994 307575 Country of ref document: US Date of ref document: 19941121 Kind code of ref document: A |
|
WWP | Wipo information: published in national office |
Ref document number: 1993907982 Country of ref document: EP |
|
WWR | Wipo information: refused in national office |
Ref document number: 1993907982 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1993907982 Country of ref document: EP |