Process for controlling contamination of oligonucleotide-dependent nucleic acid amplification reactions

Claim 2

A process for oligonucleotide-dependent amplification of one or more nucleic acid sequences in a sample, comprising the steps of: (a) amplifying nucleic acid of a first sample, wherein said amplifying is dependent on one or more specific oligonucleotides, and wherein at least one of said specific oligonucleotides comprises an exo-sample nucleotide, and (b) subjecting the nucleic acid of a second sample to treatment which renders nucleic acid containing said exo-sample nucleotide substantially unamplifiable in an amplification dependent on said specific oligonucleotides and which does not prevent amplification of nucleic acid that does not contain said exo-sample nucleotide; whereby amplified nucleic acid sequences derived from said first sample which contaminate said second sample are not further substantially amplified during amplification dependent on said specific oligonucleotides of the nucleic acid sequences of said second sample.

Claim 3

A process as claimed in Claim 1 further comprising the steps of; (c) amplifying nucleic acid sequences of said second sample.

Claim 4

A process as claimed in Claim 1 or Claim 2 further comprising the step of: (d) terminating said treatment of step (b)

Claim 5

A process as claimed in Claims 1 to 3 wherein the amplifying is a process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process further comprises: (e) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (f) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; and (g) treating the single-stranded molecules generated from step (f) with the primers of step (e) under conditions that a primer extension product is sythesized using each of the single strands produced in step (f) as a template.

Claim 6

A process for amplifying one or more nucleic acid sequences in a sample, comprising the steps of: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length: (a) treating said strands with two oligonucleotide primers, for each different specific sequence being amplified, wherein at least one primer comprises an exo-sample nucleotide, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating said single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; (d) repeating steps (a) through (c) at least once, thereby amplifying the specific nucleic acid sequence contained by said first sample; and (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences of step (A) may be present in said second sample: (e) subjecting the nucleic acid of a second sample to treatment which renders nucleic acid containing said exo-sample nucleotide substantially unamplifiable in an amplification dependent on said specific primers and which does not substantially affect amplification of nucleic acid that does not contain the exo-sample nucleotide; whereby any primer extension products of said first sample, amplified in step (A) and present in said second sample, are not further substantially amplified in step (B).

Claim 7

A process for amplifying one or more nucleic acid sequences in a sample, comprising the steps of: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length: (a) treating said strands with two oligonucleotide primers, for each different specific sequence being amplified, wherein at least one primer comprises an exo-sample nucleotide, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating said single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; (d) repeating steps (a) through (c) at least once, thereby amplifying the specific nucleic acid sequence contained by said first sample; and (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences of step (A) may be present in said second sample: (e) subjecting the nucleic acid of a second sample to treatment which renders nucleic acid containing said exo-sample nucleotide substantially unamplifiable in an amplification dependent on said specific primers and which does not substantially affect amplification of nucleic acid that does not contain the exo-sample nucleotide; whereby any primer extension products of said first sample, amplified in step (A) and present in said second sample, are not further substantially amplified in step (B).

Claim 8

A process as claimed in Claim 5, further comprising during steps (B) the step of: (f) repeating steps (a) through (c) at least once, thereby amplifying any of the specific nucleic acid sequence contained by said second sample;

Claim 9

A process as claimed in Claim 5 or Claim 6, further comprising during steps (B) the step of: (f) terminating treatment of step (e).

Claim 10

A process as claimed in Claim 4 or Claim 7, wherein the termination of the treatment is accomplished by heating.

Claim 11

A process as claimed in Claims 1-8, wherein said exo-sample nucleotide is deoxyuridine.

Claim 12

A process as claimed in Claims 1-9, wherein the treatment is selected from the group consisting of physical, chemical, enzymatic, and biological treatment.

Claim 13

A process as claimed in Claim 10, wherein said treatment is enzymatic treatment.

Claim 14

A process as claimed in Claim 11, wherein the enzymatic treatment is treatment with uracil DNA glycosylase.

Claim 15

A process as claimed in Claims 1-12, wherein at least two of said oligonucleotide primers comprise an exo-sample nucleotide.

Claim 16

A process for amplifying one or more nucleic acid sequences in a sample, comprising the steps of: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length: (a) treating said strands with two oligonucleotide primers, for each different specific sequence being amplified, wherein at least one primer comprises deoxyuridine, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for syntheis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating said single-stranded molecules generated from step (b) with the primers of step (a) under conditions that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; (d) repeating steps (a) through (c) at least once, thereby amplifying said specific nucleic acid sequence contained by the first sample; (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences of step (A) may be present in said second sample: (e) treating said strands with uracil DNA glycosylase; (f) terminating the action on the strands of the uracil DNA glycosylase by heating; and (g) repeating steps (a) through (c) at least once, thereby amplifying any of the specific nucleic acid sequence contained by second sample; whereby any primer extension products of the first sample, amplified in steps (A) and present in said second sample, are not further substantially amplified in steps (B).

Claim 17

A process for amplifying one or more nucleic acid sequences in a sample, comprising the steps of: (A) with a first sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length: (a) treating said strands with two oligonucleotide primers, for each different specific sequence being amplified, wherein at least one primer comprises deoxyuridine, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for syntheis of the extension product of the other primer; (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules; (c) treating said single-stranded molecules generated from step (b) with the primers of step (a) under conditions that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; (d) repeating steps (a) through (c) at least once, thereby amplifying said specific nucleic acid sequence contained by the first sample; (B) with a second sample containing a nucleic acid sequence or a mixture of nucleic acid sequences, wherein each nucleic acid sequence has two separate complementary strands of equal or unequal length and wherein amplified nucleic acid sequences of step (A) may be present in said second sample: (e) treating said strands with uracil DNA glycosylase; (f) terminating the action on the strands of the uracil DNA glycosylase by heating; and (g) repeating steps (a) through (c) at least once, thereby amplifying any of the specific nucleic acid sequence contained by second sample; whereby any primer extension products of the first sample, amplified in steps (A) and present in said second sample, are not further substantially amplified in steps (B).