US4994372A — DNA sequencing

Claim 1

Independent Claim 150 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in a vessel containing four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having less than 500 units of exonuclease activity per mg of said polymerase, and one of four DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each said agent terminates DNA synthesis at a different nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 2

Independent Claim 150 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in a vessel containing four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having less than 500 units of exonuclease activity per mg of said polymerase, and one of four DNA synthesis terminating agents which terminate DNA synthesis at a specific nucleotide base, wherein each said agent terminates DNA synthesis at a different nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 3

Dependent Claim 21 words

The method of claim 1 wherein said polymerase remains bound to said DNA molecule for at least 1,000 bases before dissociating.

Claim 4

Dependent Claim 14 words

The method of claim 1 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs.

Claim 5

Dependent Claim 24 words

The method of claim 1 wherein said polymerase is a modified polymerase having less than 50 units of exonuclease activity per mg of polymerase.

Claim 6

Dependent Claim 20 words

The method of claim 4 wherein said modified polymerase has less than 1 unit of activity per mg of polymerase.

Claim 7

Dependent Claim 20 words

The method of claim 4 wherein said modified polymerase has less than 0.1 unit of activity per mg of polymerase.

Claim 8

Dependent Claim 13 words

The method of claim 1 wherein said polymerase has no detectable exonuclease activity.

Claim 9

Dependent Claim 19 words

The method of claim 1 wherein said polymerase is able to utilize primers of 10 base pairs or more.

Claim 10

Dependent Claim 19 words

The method of claim 1 wherein said polymerase is able to utilize primers of 4 base pairs or more.

Claim 11

Dependent Claim 24 words

The method of claim 1 wherein said primer comprises 4-20 base pairs and said polymerase is able to utilize primers of 4-20 base pairs.

Claim 12

Dependent Claim 14 words

The method of claim 1 wherein said primer is single stranded DNA or RNA.

Claim 13

Dependent Claim 15 words

The method of claim 1 wherein said incubating comprises a pulse and a chase step.

Claim 14

Dependent Claim 38 words

The method of claim 12 wherein said pulse step comprises mixing said annealed molecules with all four deoxynucleoside triphosphates and a processive DNA polymerase, wherein at least one said deoxynucleoside triphosphate is labelled and present in limiting concentration.

Claim 15

Dependent Claim 19 words

The method of claim 13 wherein said pulse step incubation is carried out for 30 seconds to 20 minutes.

Claim 16

Dependent Claim 33 words

The method of claim 13 wherein said chase step comprises adding a different one of said chain terminating agents to each of four separate aliquots of the mixture after performing said pulse step.

Claim 17

Dependent Claim 18 words

The method of claim 15 wherein said chase step incubation is carried out for 1 to 60 minutes.

Claim 18

Dependent Claim 13 words

The method of claim 1 wherein said terminating agent is a dideoxynucleoside triphosphate.

Claim 19

Dependent Claim 18 words

The method of claim 1 wherein one said deoxynucleoside triphosphate is chosen from deoxyinosine triphosphate or deazaguanosine triphosphate.

Claim 20

Dependent Claim 10 words

The method of claim 1 wherein said primer is labelled.

Claim 21

Dependent Claim 12 words

The method of claim 19 wherein said incubating comprises a chase step.

Claim 22

Dependent Claim 11 words

The method of claim 19 wherein said primer is fluorescently labelled.

Claim 23

Dependent Claim 68 words

A kit for DNA sequencing, comprising: a processive DNA polymerase, said polymerase having less than 500 units of exonuclease activity per mg of polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) dITP and (b) a chain terminating agent.

Claim 24

Dependent Claim 68 words

A kit for DNA sequencing, comprising: a processive DNA polymerase, said polymerase having less than 500 units of exonuclease activity per mg of polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) dITP and (b) a chain terminating agent.

Claim 25

Dependent Claim 30 words

The method of claim 1, wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequencing reaction.

Claim 26

Dependent Claim 30 words

The kit of claim 22 wherein said polymerase is unable to exhibit its processivity in a second environmental condition normally used in the pulse step of a DNA sequencing reaction.

Claim 27

Dependent Claim 13 words

The kit of claim 22 in which said reagent is a dideoxynucleoside triphosphate.

Claim 28

Independent Claim 142 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in the presence of four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 29

Independent Claim 142 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in the presence of four different deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 30

Dependent Claim 27 words

The method of claim 26 wherein said polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

Claim 31

Dependent Claim 15 words

The method of claim 26 wherein said processive DNA polymerase is a T7-type DNA polymerase.

Claim 32

Dependent Claim 29 words

The method of claim 28 wherein said T7-type DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

Claim 33

Dependent Claim 14 words

The method of claim 28 wherein said T7-type DNA polymerase is T7 DNA polymerase.

Claim 34

Dependent Claim 29 words

The method of claim 28 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

Claim 35

Dependent Claim 90 words

A kit for DNA sequencing, comprising: a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) deoxyinosine triphosphate and (b) a chain terminating agent.

Claim 36

Dependent Claim 90 words

A kit for DNA sequencing, comprising: a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having less than 50% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) deoxyinosine triphosphate and (b) a chain terminating agent.

Claim 37

Dependent Claim 27 words

The kit of claim 32 wherein said polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

Claim 38

Dependent Claim 15 words

The kit of claim 32 wherein said processive DNA polymerase is a T7-type DNA polymerase.

Claim 39

Dependent Claim 29 words

The kit of claim 34 wherein said T7-type DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase.

Claim 40

Dependent Claim 14 words

The kit of claim 34 wherein said T7-type DNA polymerase is T7 DNA polymerase.

Claim 41

Dependent Claim 28 words

The kit of claim 36 wherein said T7 DNA polymerase has less than 1% of the exonuclease activity of the naturally-associated level of exonuclease activity of said polymerase.

Claim 42

Independent Claim 145 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in the presence of four deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 43

Independent Claim 145 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed molecules in the presence of four deoxynucleoside triphosphates, a processive DNA polymerase, wherein said polymerase is able to remain bound for at least 500 bases to said DNA molecule in an environmental condition used in the extension reaction of a DNA sequencing reaction, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base, and separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 44

Dependent Claim 23 words

The method of claim 1 or 38 wherein said annealed molecules are incubated in at least four vessels containing four different deoxynucleoside triphosphates.

Claim 45

Dependent Claim 93 words

A kit for DNA sequencing, comprising: a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) deoxyinosine triphosphate (b) a chain terminating agent.

Claim 46

Dependent Claim 93 words

A kit for DNA sequencing, comprising: a processive DNA polymerase which remains bound to a DNA molecule for at least 500 bases before dissociating, said polymerase having a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined, said polymerase being able to exhibit its processivity in an environmental condition normally used in the extension reaction of a DNA sequencing reaction, and a reagent necessary for said sequencing, selected from the group consisting of (a) deoxyinosine triphosphate (b) a chain terminating agent.

Claim 47

Independent Claim 108 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed primer molecule and the DNA molecule in the presence of at least four deoxynucleotide triphosphates, a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base; separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 48

Independent Claim 108 words

A method for determining the nucleotide base sequence of a DNA molecule, comprising: providing said DNA molecule annealed with a primer molecule able to hybridize to said DNA molecule; incubating the annealed primer molecule and the DNA molecule in the presence of at least four deoxynucleotide triphosphates, a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of said polymerase, and a DNA synthesis terminating agent which terminates DNA synthesis at a specific nucleotide base; separating the DNA products of the incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.

Claim 49

Dependent Claim 13 words

The method of claim 41 wherein said DNA polymerase is T7 DNA polymerase.

Claim 50

Dependent Claim 37 words

A kit for DNA sequencing, comprising: a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, and a reagent selected from the group consisting of dITP, and a chain terminating agent.

Claim 51

Dependent Claim 37 words

A kit for DNA sequencing, comprising: a T7-type DNA polymerase having less than 500 units of exonuclease activity per mg of polymerase, and a reagent selected from the group consisting of dITP, and a chain terminating agent.

Claim 52

Dependent Claim 13 words

The kit of claim 43 wherein said DNA polymerase is T7 DNA polymerase.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1994-02-18	1994-08-20	1995-02-19	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1998-02-18	1998-08-20	1999-02-19	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2002-02-18	2002-08-20	2003-02-19	$7700.00	Window Closed / Lapsed

DNA sequencing

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