Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1998-01-09
2000-09-26
Jones, W. Gary
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435183, 536 231, 536 253, 536 254, C12Q 168
Patent
active
061240931
DESCRIPTION:
BRIEF SUMMARY
Subject matter of the invention is a method of transcribing ribonucleic acids (RNA) of a sample into deoxyribonucleic acids (DNA); further, a method of preparing a multitude of DNA molecules with the aid of RNA molecules, reagents for reducing artifacts and reagent kits for implementing said method.
Methods of analyzing nucleic acids can be based on the analysis of both RNA and DNA. Both variants have their advantages. In methods that are based on the analysis of RNA, the ribonucleic acid is first transcribed into deoxyribonucleic acid, the reason being that deoxyribonucleic acids are more stable. However, ribonucleic acids often provide information which is not available when DNA is used, because genomic DNA contains intron sequences that are lost when the RNA is processed by the organism. Methods that are based on the analysis of DNA can, therefore, yield the same result depending on whether or not the sequence to be analyzed contains introns. In cases where only the RNA is to be detected, contamination by DNA leads to a positive alteration of the results unless certain precautions are taken. One possibility of solving this problem is the use of junction primers. Junction primers are oligonucleotides whose sequence is selected such that the 5'-end thereof is complementary to the sequence of the 5'-end of an exon while the 3'-end contains nucleotides that are complementary to the nucleotides of the 3'-end of the adjacent exon but not to the nucleotides of the 3'-end of the intron located between the two in the genome. In this case, the alteration of the result by contaminating DNA can at least be partly avoided.
However, such a distinction cannot be made between DNA and RNA when analyzing transcripts of an intron-free gene. Moreover, pseudogenes pose the same problem as they are identical in length and therefore cannot be distinguished from RNA transcripts. In such cases, it has proven to be advantageous to treat the mRNA sample with RNase-free DNase prior to the transcription reaction. A method of this kind has been described in BioTechniques 9: 262-268 (1990) and BioTechniques 13: 726-729 (1992).
The evaluation of effective DNA digestion when dealing with gene portions that contain introns requires that a part of the RNA that was treated with DNase but not reversely transcribed be subject to a polymerase chain reaction using a primer set which allows discrimination of PCR products according to size that are a result of the cDNA or the genomic DNA.
DNase I requires divalent metal ions (J. Biol. Chem. 243: 4409-4416 (1968)) as a cofactor. It is known that an optimum Mg.sup.2 +ion concentration is one which exceeds the Mn.sup.2+ concentration by one order of magnitude. Moreover, it is also known that an increase in the Mn.sup.2+ concentration does not inhibit the DNase reaction as is the case with Mg.sup.2+ or Ca.sup.2+.
Core of the present invention is the finding that the use of Mn.sup.2+ ions significantly reduces or even completely eliminates the amount of artifact DNA formed by replicative amplification, e.g. by means of the polymerase chain reaction (PCR). Moreover, DNase digestion with Mn.sup.2+ ions is considerably more effective than with Mg.sup.2+.
Subject matter of the present invention is therefore a method of transcribing ribonucleic acids of a sample into deoxyribonucleic acids, comprising the following steps: enzyme, followed by
Other subject matters of the invention are a method of preparing a multitude of DNA molecules with the aid of RNA molecules, reagent kits for use in said method, and the use of Mn.sup.2+ ions to reduce the formation of artifact DNA in transcription reactions.
A ribonucleic acid for use in the present invention can be any desired ribonucleic acid, e.g. mRNA, tRNA, rRNA. The invention works particularly well with mRNA that contains no introns and mRNA that is present as a pseudogene or about which it is not known whether pseudogenes are present or not.
In accordance with the invention, ribonucleic acids can be present even when highly contaminated with corresponding DN
REFERENCES:
patent: 5561058 (1996-10-01), Gelfland et al.
patent: 5843686 (1998-12-01), Zain et al.
Myers et al. "Reverse Transcription and DNA Amplification . . . " Biochemistry. vol. 30, No. 31. pp. 7661-7666, Aug. 6, 1991.
A. Rolfs et al., Method for Identification of Amplified PCR Products, "PCR: Clinical Diagnostics and Research".
Biotechniques, vol. 13, No. 5, (1992), "Single-Cell cDNA-PCR: Removal of Contaminating Genomic DNA from Total RNA Using Immobilized Dnase I".
Biotechniques, vol. 9, No. 3, (1996), "Use of Reverse Transcriptase Polymerase Chain Reaction to Monitor Expression of Intronless Genes".
Melgar et al., The Journal of Biological Chemistry, vol. 243, No. 17, "Deoxyribonucleic Acid Nucleases".
Deragon et al., "Nucleic Acids Research", 18 (1990) Oct. 25, No. 20, "Use of y irradiation to eliminate DNA contamination for PCR".
Bauer Peter
Fleck Eckart
Regitz-Zagrosek Vera
Rolfs Arndt
Jones W. Gary
Roche Diagnostics GmbH
Taylor Janell E.
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