Amplification of nucleic acid sequences using oligonucleotides of random sequences as primers

• US 5,106,727 A
• Filed: 07/13/1990
• Issued: 04/21/1992
• Est. Priority Date: 04/27/1989
• Status: Expired due to Term

• Current Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Hartley, James L., Berninger, Mark S.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/552,407
• Time in Patent Office: 648 Days
• Field of Search: 435/5, 435/6, 435/91, 435/94, 436/501, 536/26, 536/27, 536/28, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• 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 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

According to this invention, a process for substantially amplifying template nucleic acid present in a sample is described, wherein said amplification may be performed without prior knowledge of specific sequences, which process comprises apposition of random oligonucleotide primers to said template nucleic acid under conditions such that extension products of said primers are synthesized which are complementary to said template nucleic acid.

Claims

1. A method for reducing de novo amplification in a process for substantially amplifying template nucleic acid sequences in a sample comprising amplification of nucleic acid in a randomly primed and template dependent manner, wherein the randomly primed amplification occurs with an excess of random oligonucleotide primers that comprise ribonucleotides or deoxyribonucleotides having bases chosen from the group of A, T, C, and I,

wherein the base U and the base T are considered exact equivalents,

whereby de novo amplification is reduced relative to the level observed with random oligonucleotide primers comprising bases chosen from the group A, T, C, and G.

2. A method for reducing de novo amplification in a process for substantially amplifying template nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising the steps of:

(a) priming template nucleic acid strands with an excess of random oligonucleotide primers; and

(b) incubating said template nucleic acid strands and said excess random oligonucleotide primers in the presence of an excess of an inducing agent, a strand displacement agent, and an excess of triphosphate substrates to randomly amplify nucleic acid strands, wherein the random oligonucleotide primers comprise ribonucleotides or deoxyribonucleotides having bases chosen from the group of A, T, C, and I,

wherein the base U and the base T are considered exact equivalents,

whereby de novo amplification is reduced relative to the level observed with random primers comprising bases A, T, C, and G.

3. A method for reducing de novo amplification in a process for substantially amplifying template nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising the steps of:

(a) priming template nucleic acid strands with an excess of random oligonucleotide primers wherein said primers consist of 60-mers and 10-mers; and

(b) incubating said template nucleic acid strands and said excess random oligonucleotide primers in the presence of an excess of the Klenow fragment of DNA Polymerase I and an excess of triphosphate substrates to randomly amplify nucleic acid strands, wherein the random oligonucleotide primers comprise ribonucleotides or deoxyribonucleotides having bases chosen from the group of A, T, C, and I,

wherein the base U and the base T are considered exact equivalents,

whereby de novo amplification is reduced relative to the level observed with random primers comprising bases A, T, C, and G.

4. A method of reducing de novo amplification in a process for detecting a papilloma virus in a sample comprising:

(a) substantial amplification of said papilloma virus nucleic acid sequences in said sample wherein said substantial amplification comprises a randomly primed but template dependent synthesis of papilloma virus DNA; and

(b) detecting said papilloma virus, wherein the random oligonucleotide primers comprise an excess of ribonucleotides or deoxyribonucleotides having bases chosen from the group of A, T, C, and I,

wherein the base U and the base T are considered exact equivalents,

whereby de novo amplification is reduced relative to the level observed with random primers comprising bases A, T, C, and G.

5. A kit for reducing de novo amplification while substantially amplifying nucleic acid sequences in a sample in a randomly primed and template dependent manner, comprising a carrier being compartmentalized to receive in close confinement therein one or more containers wherein:

(a) a first container or series of containers contains random oligonucleotide primers;

(b) a second container contains an inducing agent;

(c) a third container or series of containers contains triphosphate substrates; and

(d) a fourth container or series of containers contains buffer for reconstituting or diluting components of said kit, wherein the random oligonucleotide primers comprise ribonucleotides or deoxyribonucleotides having bases chosen from the group of A, T, C, and I,

wherein the base U and the base T are considered exact equivalents,

whereby de novo amplification is reduced relative to the level observed with random primers comprising bases A, T, C, and G.