WO1997020949A1 - An improved method for dye-terminator (cycle) sequencing - Google Patents

An improved method for dye-terminator (cycle) sequencing Download PDF

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Publication number
WO1997020949A1
WO1997020949A1 PCT/GB1996/003041 GB9603041W WO9720949A1 WO 1997020949 A1 WO1997020949 A1 WO 1997020949A1 GB 9603041 W GB9603041 W GB 9603041W WO 9720949 A1 WO9720949 A1 WO 9720949A1
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Prior art keywords
sequencing
terminator
cycle
dye
deaza
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PCT/GB1996/003041
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French (fr)
Inventor
Michael Alan Reeve
Philip Steven Robinson
Helen Franklin
Bernie Mcardle
Carl Fuller
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Amersham International Plc
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Publication of WO1997020949A1 publication Critical patent/WO1997020949A1/en

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    • 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

  • DNA sequencing relies upon the synthesis of a new strand of DNA starting at a specific priming site and ending with the inco ⁇ oration of a chain terminating nucleotide such as a dideoxynucleoside triphosphate
  • Cycle sequencing (2) uses repeated cycles of thermal denaturation, annealing and extension/termination to increase signal levels and therefore decrease the amount of template required. For each cycle, the amount of product DNA will be equivalent to the amount of primed template. However, if a thermostable enzyme such as Thermo SequenaseTM (3) is used, many cycles can be performed without the need to add fresh enzyme. For example, after ten cycles there could be up to ten times as much product as template (in practice, each cycle is not 100% efficient and the amplification is somewhat lower than this).
  • Cycle sequencing is therefore ideally suited for applications where the amount of template may be limiting or where the sensitivity of the detection system is not high.
  • dye-terminator (cycle) sequencing refers to dye-terminator sequencing whether cycle sequencing (2) or noncycle sequencing (the latter being sometimes referred to as 'unicycle' sequencing) ln fluorescent dye-terminator (cycle) sequencing, the label is incorporated on the chain terminating nucleotide.
  • cycle sequencing (2) or noncycle sequencing the latter being sometimes referred to as 'unicycle' sequencing
  • cycle sequencing (3) sequencing the latter being sometimes referred to as 'unicycle' sequencing
  • ln fluorescent dye-terminator (cycle) sequencing the label is incorporated on the chain terminating nucleotide.
  • the relative concentrations of dNTPs and dye-terminators are balanced so that, on average, at each position within the desired length of sequence, there is an equal chance of a dye-terminator being incorporated.
  • sequence information can be obtained.
  • 7-deaza-dGTP and dITP have an influence on the choice between: dA and the A-terminator; dCTP and the C-terminator; dTTP and the T-terminator as well as 7-deaza-dGTP or dITP and the G-terminator.
  • dye-terminator (cycle) sequencing with dITP requires long extension times in the (cycle) sequencing. 4 minute extension times are required, rather than 30 second extension times that are customary for dye-terminator (cycle) sequencing with dGTP.
  • the Current Invention describes use of a number of dNTP analogues.
  • the inclusion of these dNTP analogues in DNA sequencing reactions leads to an improvement of the peak height smoothness when compared to dITP or 7-deaza-dGTP sequencing.
  • the above analogues give more even peak heights; resulting in several advantages: it is possible to read a longer sequence; it is possible to identify individual bases more accurately and reliably; it makes heterozygote analysis with dye-terminator (cycle) sequencing a less difficult task.
  • 0.5 ⁇ g of pPR26 plasmid template were cycle sequenced using the Universal Sequencing Primer (US70706, Amersham International pic) on a TMVistra DNA Systems DNA Labstation 625 (Amersham International pic).
  • the Labstation Thermo SequenaseTM fluorescent dye- terminator cycle sequencing kit (RPN2435, Amersham International pic) and the Labstation FMPTM fluorescent dye-terminator precipitation kit (RPN2433, Amersham International pic) were used as supplied except where stated below.
  • the 1-32 dye-terminator cycle sequencing v2.0 method was used as supplied.
  • dITP was used at a final concentration of 300 ⁇ M in the sequencing reaction.
  • 7-deaza-dlTP was used at a final concentration of 100 ⁇ M in the sequencing reaction.
  • pBluescript plasmid template 0.5 ⁇ g of pBluescript plasmid template were cycle sequenced using the Reverse Sequencing Primer (US71803, Amersham International pic) on a TMVistra DNA Systems DNA Labstation 625 (Amersham International pic).
  • the Labstation Thermo SequenaseTM fluorescent dye- terminator cycle sequencing kit (RPN2435, Amersham International pic) and the Labstation FMPTM fluorescent dye-terminator precipitation kit (RPN2433, Amersham International pic) were used as supplied except where stated below.
  • the 1 -32 dye-terminator cycle sequencing v2.0 method was used as supplied.
  • dATP was used at a final concentration of 100 ⁇ M in the sequencing reaction.
  • 7-deaza-dATP was used at a final concentration of 100 ⁇ M in the sequencing reaction.
  • the samples were then electrophoresed and analysed on an Applied Biosystems 373 fluorescent sequencer.

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Abstract

An improved method for dye-terminator(cycle) sequencing involves using dNTP analogues which lead to an improvement in the peak height smoothness of a sequence print-out. The analogues are 7-deaza-dITP used in place of dGTP, 7-deaza-dATP used in place of dATP, and 4-methyl-dCTP used in place of dCTP.

Description

AN IMPROVED METHOD FOR DYE-TERMINATOR (CYCLE)
SEQUENCING
INTRODUCTION: DNA sequencing
DNA sequencing relies upon the synthesis of a new strand of DNA starting at a specific priming site and ending with the incoφoration of a chain terminating nucleotide such as a dideoxynucleoside triphosphate
(1).
Cycle sequencing
Cycle sequencing (2) uses repeated cycles of thermal denaturation, annealing and extension/termination to increase signal levels and therefore decrease the amount of template required. For each cycle, the amount of product DNA will be equivalent to the amount of primed template. However, if a thermostable enzyme such as Thermo Sequenase™ (3) is used, many cycles can be performed without the need to add fresh enzyme. For example, after ten cycles there could be up to ten times as much product as template (in practice, each cycle is not 100% efficient and the amplification is somewhat lower than this).
Cycle sequencing is therefore ideally suited for applications where the amount of template may be limiting or where the sensitivity of the detection system is not high.
Dye-Terminator (Cycle) Sequencing:
The term dye-terminator (cycle) sequencing mentioned herein, refers to dye-terminator sequencing whether cycle sequencing (2) or noncycle sequencing (the latter being sometimes referred to as 'unicycle' sequencing) ln fluorescent dye-terminator (cycle) sequencing, the label is incorporated on the chain terminating nucleotide. The relative concentrations of dNTPs and dye-terminators are balanced so that, on average, at each position within the desired length of sequence, there is an equal chance of a dye-terminator being incorporated.
When these fragments are separated on a suitable gel matrix, sequence information can be obtained.
The Current Art: Dye-terminator (cycle) sequencing with thermostable polymerases and the ABI Dye-Deoxy™ reagents suffers from one clear flaw.
During electrophoresis, certain sequences (the sequence 5'-dG-dC-ddT-TMR-3', where TMR denotes tetramethylrhodamine is one particular example) exhibit a phenomenon often referred to as 'dye- terminator compression'. As a result of 'dye-terminator compression', the above sequence runs with the peak order GTC, rather than the expected GCT.
Replacement of the dG residue in this sequence with either dl or 7-deaza-dG (achieved by the replacement of dGTP by either dITP or 7- deaza-dGTP in the sequencing reaction) reverts the peak order from the incorrect GTC to the expected GCT.
Replacement of dGTP by either dITP or 7-deaza-dGTP in the sequencing reaction, however, leads to a marked deterioration of the peak height smoothness seen in the sequence. The observed peak height unevenness is sequence specific. This unevenness occurs despite the fact that for every 'decision' by the polymerase, the ratio of dNTP to its cognate ddNTP-dye is virtually the same. The inclusion of 7-deaza-dGTP or dITP to the dNTPs mix has an effect on the peak height unevenness for all four bases, i e. 7-deaza-dGTP and dITP have an influence on the choice between: dA and the A-terminator; dCTP and the C-terminator; dTTP and the T-terminator as well as 7-deaza-dGTP or dITP and the G-terminator.
In addition, dye-terminator (cycle) sequencing with dITP requires long extension times in the (cycle) sequencing. 4 minute extension times are required, rather than 30 second extension times that are customary for dye-terminator (cycle) sequencing with dGTP.
The Current Invention: The current invention describes use of a number of dNTP analogues. The inclusion of these dNTP analogues in DNA sequencing reactions leads to an improvement of the peak height smoothness when compared to dITP or 7-deaza-dGTP sequencing.
Three analogues will be described: 7-deaza-dlTP (used in place of dGTP), 7-deaza-dATP (used in place of dATP) and 4-methyl- dCTP (used in place of dCTP).
The above analogues give more even peak heights; resulting in several advantages: it is possible to read a longer sequence; it is possible to identify individual bases more accurately and reliably; it makes heterozygote analysis with dye-terminator (cycle) sequencing a less difficult task.
In general, use of the three analogues is found to improve peak height smoothness by about 5-10%.
EXAMPLES:
Example 1
7-deaza-dlTP (fig.1) versus dITP (fig. 2)
0.5 μg of pPR26 plasmid template were cycle sequenced using the Universal Sequencing Primer (US70706, Amersham International pic) on a ™Vistra DNA Systems DNA Labstation 625 (Amersham International pic). The Labstation Thermo Sequenase™ fluorescent dye- terminator cycle sequencing kit (RPN2435, Amersham International pic) and the Labstation FMP™ fluorescent dye-terminator precipitation kit (RPN2433, Amersham International pic) were used as supplied except where stated below. The 1-32 dye-terminator cycle sequencing v2.0 method was used as supplied. dITP was used at a final concentration of 300 μM in the sequencing reaction. 7-deaza-dlTP was used at a final concentration of 100 μM in the sequencing reaction.
The samples were then electrophoresed and analysed on an Applied Biosystems 373 fluorescent sequencer.
Example 2
7-deaza-dATP (fig. 3) versus dATP (fig. 4)
0.5 μg of pBluescript plasmid template were cycle sequenced using the Reverse Sequencing Primer (US71803, Amersham International pic) on a TMVistra DNA Systems DNA Labstation 625 (Amersham International pic). The Labstation Thermo Sequenase™ fluorescent dye- terminator cycle sequencing kit (RPN2435, Amersham International pic) and the Labstation FMP™ fluorescent dye-terminator precipitation kit (RPN2433, Amersham International pic) were used as supplied except where stated below. The 1 -32 dye-terminator cycle sequencing v2.0 method was used as supplied. dATP was used at a final concentration of 100 μM in the sequencing reaction.
7-deaza-dATP was used at a final concentration of 100 μM in the sequencing reaction. The samples were then electrophoresed and analysed on an Applied Biosystems 373 fluorescent sequencer.
Example 3
4-methyl-dCTP (fig. 5) versus dCTP (Fig. 6) 0.5 μg of M13mp8 template were cycle sequenced using the
Universal Sequencing Primer (US70706, Amersham International pic) on a ™Vistra DNA Systems DNA Labstation 625 (Amersham International pic). The Labstation Thermo Sequenase™ fluorescent dye-terminator cycle sequencing kit (RPN2435, Amersham International pic) and the Labstation FMP™ fluorescent dye-terminator precipitation kit (RPN2433, Amersham International pic) were used as supplied except where stated below. The 1-32 dye-terminator cycle sequencing v2.0 method was used as supplied. dCTP was used at a final concentration of 100 μM in the sequencing reaction. 4-methyl-dCTP was used at a final concentration of 200 μM in the sequencing reaction.
The samples were then electrophoresed and analysed on an Applied Biosystems 373 fluorescent sequencer.
REFERENCES:
1. Sanger, F., Niklen, S. and Coulson, A.R., Proc. Nat. Acad. Sci. (USA), 74, pp 5463-5467, (1977) .
2. Murray, V. er al., Nucleic Acids Research, 17, p8889, (1989).
3. Reeve, M. A. and Fuller, C. W., Nature, 376, pp796-797, (1995).

Claims

1. A method for dye-terminator (cycle) sequencing wherein dNTP analogues are used which lead to an improvement of the peak height smoothness.
2. A method for dye-terminator (cycle) sequencing as in claim 1 wherein 7-deaza-dlTP is used in place of dGTP in the sequencing reaction.
3. A method for dye-terminator (cycle) sequencing as in claim 1 wherein 7-deaza-dATP is used in place of dATP in the sequencing reaction.
4. A method for dye-terminator (cycle) sequencing as in claim 1 wherein 4-methyl-dCTP is used in place of dCTP in the sequencing reaction.
5. A method for dye-terminator (cycle) sequencing as in claim 1 wherein any of the analogues in claims 2 to 4 are used in any combination in the sequencing reaction.
6. A DNA (cycle) sequencing kit for use in the method of any one of claims 1 to 5, comprising a supply of nucleotides or nucleotide analogues including at least one of 7-deaza-dlTP, 7-deaza-dATP and 4-methyl-dCTP, a supply of labelled chain-terminating nucleotide analogues, a primer and a polymerase enzyme.
PCT/GB1996/003041 1995-12-06 1996-12-06 An improved method for dye-terminator (cycle) sequencing WO1997020949A1 (en)

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GB9524974.4 1995-12-06

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041410A1 (en) * 1998-02-10 1999-08-19 Yeda Research And Development Co. Ltd. Methods for dna amplification and sequencing
FR2778414A1 (en) * 1998-04-24 1999-11-12 Visible Genetics Inc PROCESS FOR SEQUENCING POLYMERS OF NUCLEIC ACIDS
WO2001002602A2 (en) * 1999-07-05 2001-01-11 Lion Bioscience Ag Nucleotides for cycle sequencing of gc-rich templates
US6605434B1 (en) 1999-03-16 2003-08-12 Human Genome Sciences, Inc. Direct bacterial lysate sequencing

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011533A1 (en) * 1990-01-26 1991-08-08 E.I. Du Pont De Nemours And Company Method for isolating primer extension products from template-directed dna polymerase reactions
WO1994005684A1 (en) * 1992-09-04 1994-03-17 Life Technologies, Inc. N4-methyl-2'-deoxycytidine 5'-triphosphate and its use in polymerase-catalyzed nucleic acid syntheses
WO1994023055A1 (en) * 1993-03-30 1994-10-13 United States Biochemical Corporation Cycle sequencing with non-thermostable dna polymerases
WO1994023066A1 (en) * 1993-03-30 1994-10-13 United States Biochemical Corporation Use of exonuclease in dna sequencing
EP0655506A1 (en) * 1994-10-17 1995-05-31 President And Fellows Of Harvard College DNA polymerases having modified nucleotide binding site for DNA sequencing

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991011533A1 (en) * 1990-01-26 1991-08-08 E.I. Du Pont De Nemours And Company Method for isolating primer extension products from template-directed dna polymerase reactions
WO1994005684A1 (en) * 1992-09-04 1994-03-17 Life Technologies, Inc. N4-methyl-2'-deoxycytidine 5'-triphosphate and its use in polymerase-catalyzed nucleic acid syntheses
WO1994023055A1 (en) * 1993-03-30 1994-10-13 United States Biochemical Corporation Cycle sequencing with non-thermostable dna polymerases
WO1994023066A1 (en) * 1993-03-30 1994-10-13 United States Biochemical Corporation Use of exonuclease in dna sequencing
EP0655506A1 (en) * 1994-10-17 1995-05-31 President And Fellows Of Harvard College DNA polymerases having modified nucleotide binding site for DNA sequencing

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999041410A1 (en) * 1998-02-10 1999-08-19 Yeda Research And Development Co. Ltd. Methods for dna amplification and sequencing
US6428986B1 (en) 1998-02-10 2002-08-06 Yeda Research And Development Co., Ltd. At The Weizmann Institute Of Science Methods for DNA amplificating and sequencing
US7150980B1 (en) 1998-02-10 2006-12-19 Yeda Research And Development Co. Ltd. Methods for DNA amplification and sequencing
US7387878B2 (en) 1998-02-10 2008-06-17 Yeda Research And Development Co. Ltd. Methods for DNA amplification and sequencing
FR2778414A1 (en) * 1998-04-24 1999-11-12 Visible Genetics Inc PROCESS FOR SEQUENCING POLYMERS OF NUCLEIC ACIDS
US6605434B1 (en) 1999-03-16 2003-08-12 Human Genome Sciences, Inc. Direct bacterial lysate sequencing
WO2001002602A2 (en) * 1999-07-05 2001-01-11 Lion Bioscience Ag Nucleotides for cycle sequencing of gc-rich templates
EP1074633A1 (en) * 1999-07-05 2001-02-07 LION Bioscience AG Nucleotides for cycle sequencing of gc-rich templates
WO2001002602A3 (en) * 1999-07-05 2001-07-05 Lion Bioscience Ag Nucleotides for cycle sequencing of gc-rich templates

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