Process for controlling contamination of nucleic acid amplification reactions

A process for oligonucleotide-dependent amplification of one or more nucleic acid molecules in a sample, comprising the steps of:

• (a) amplifying nucleic acid era first sample, wherein said amplifying is dependent on one or more oligonucleotide primers, and wherein at least one of said oligonucleotide primers comprises one or more deoxyuridine nucleotides at or near at least one of its termini, thereby producing amplified nucleic acid containing deoxyuridine;
• (b) subjecting a second sample comprising nucleic acid to an enzymatic treatment with uracil DNA glycosylase, which renders said amplified nucleic acid containing deoxyuridine obtained in step (a), and contaminating said second sample, substantially unamplifiable and wherein the uracil DNA glycosylase treatment does not substantially prevent oligonucleotide-dependent amplification of a nucleic acid that does not contain deoxyuridine;
• (c) amplifying the nucleic acid of said second sample;
• and
• (d) terminating said treatment of step (b) by heating.

A process for oligonucleotide-dependent amplification of one or more nucleic acid molecules in a sample, comprising the steps of:

• (a) amplifying nucleic acid of a first sample, wherein said amplifying is dependent on one or more oligonucleotide primers, and wherein at least one of said oligonucleotide primers comprises one or more deoxyuridine nucleotides, thereby producing amplified nucleic acid containing deoxyuridine;
• (b) subjecting a second sample comprising nucleic acid to an enzymatic treatment with uracil DNA glycosylase which renders said amplified nucleic acid containing deoxyuridine obtained in step (a), and contaminating said second sample, substantially unamplifiable and wherein said enzymatic treatment does not substantially prevent oligonucleotide-dependent amplification of a nucleic acid that does not contain deoxyuridine;
• (c) amplifying the nucleic acid of said second sample;
• and
• (d) terminating said treatment of step (b) by heating.

A method of making a DNA molecule comprising the steps of:

• (a) treating a nucleic acid strand with an oligonucleotide primer, wherein said primer comprises one or more exo-sample nucleotides, under conditions such that an extension product is synthesized which is complementary to said nucleic acid strand, and
• (b) subjecting the extension product of step (a) to a treatment which removes the base of said exo-sample nucleotide;
• whereby at least one apyrimidinic deoxyribose in the sugar-phosphate backbone of said extension product is formed;
• and
• (c) subjecting the extension product containing an apyrimidinic deoxyribose obtained in step (b) to a treatment which cleaves said sugar-phosphate backbone to give said DNA molecule.

A method of making a DNA molecule comprising the steps of:

• (a) treating a nucleic acid strand with an oligonucleotide primer, wherein said primer comprises one or more deoxyuridine nucleotides, under conditions such that an extension product is synthesized which is complementary to said nucleic acid strand;
• (b) subjecting the extension product of step (a) to enzymatic treatment with uracil DNA glycosylase which removes the base of said exo-sample nucleotide;
• whereby at least one apyrimidinic deoxyribose in the sugar-phosphate backbone of said extension product is formed;
• and
• (c) subjecting the extension product containing an apyrimidinic deoxyribose obtained in step (b) to a treatment which cleaves said sugar-phosphate backbone to give said DNA molecule, wherein said treatment is either alkaline hydrolysis or enzymatic treatment with endonuclease IV.