Process for controlling contamination of nucleic acid amplification reactions

• US 5,035,996 A
• Filed: 06/01/1989
• Issued: 07/30/1991
• Est. Priority Date: 06/01/1989
• Status: Expired due to Term

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated), Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Steffe, Eric
• Application Number:
  US07/360,120
• Time in Patent Office: 789 Days
• Field of Search: 435/6, 435/91, 435/227, 435/200, 536/21
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

In the process according to this invention, an amplification procedure is performed on a first sample in which one or more of the four normal ribonucleoside triphosphates (rNTPs) or deoxyribonucleoside triphosphates (dNTPs) is replaced with an exo-sample nucleotide. After amplification, any contaminating amplified product that may be remaining is subjected to a physical, chemical, enzymatic, or biological treatment which renders nucleic acid containing the exo-sample nucleotide substantially unamplifiable. The treatment may be done as a separate step or it may be done in the presence of a second sample containing nucleic acid sequences to be amplified. The amplified nucleic acid sequences derived from the first sample which contaminate the second sample are not further substantially amplified during amplification of nucleic acid sequences of the second sample.

Claims

1. A method for controlling contamination in sequential nucleic acid amplification processes comprising first and second nucleic acid amplification processes to amplify nucleic acid sequences in a first and a second sample, respectively, which comprises:

carrying out said first nucleic acid amplification process on said nucleic acid sequence in said first sample in the presence of deoxyuridine triphosphate, to thereby generate a deoxyuracil-containing product of amplification; and

treating said deoxyuracil-containing product of amplification with uracil DNA glycosylase prior to carrying out said second amplification process on said nucleic acid sequence in said second sample.

2. The process of claim 1 wherein said nucleic acid sequences in said first and second samples have two separate complementary strands each,

wherein said first nucleic acid amplification process on said first sample comprises:

(1) treating said 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 and which incorporates deoxyuridine, 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;

(2) separating the primer extension products from the templates on which they are synthesized to produce single-stranded molecules;

(3) treating said single-stranded molecules generated from step (2) with the primers of step (1) under conditions that a primer extension product is synthesized using each of the single strands produced in step (2) as a template;

(4) repeating steps (1) through (3) at least once, thereby amplifying said specific nucleic acid sequence contained in said first sample;

and wherein said second nucleic acid amplification process on said second sample comprises

(5) treating said second sample with uracil DNA glycosylase; and

(6) repeating steps (1) through (3) on said second sample at least once, thereby amplifying the nucleic acid sequence contained in said second sample.

3. The process of claim 1 wherein, after treating said deoxyuracil-containing product with uracil DNA glycosylase, and prior to carrying out said second amplification process, the second sample is heated so as to terminate the action of said uracil DNA glycosylase.

4. The process of claim 2 wherein, after said step (5), the process comprises:

(7) heating said sample so as to terminate the action of said uracil DNA glycosylase.