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WO 2011/010094 Al (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 27 January 2011 (27.01.2011) WO 2011/010094 Al (51) International Patent Classification: (74) Agents: GARDNER, Rebecca et al; Dehns, St Bride's C12Q 1/68 (2006.0 1) C12N 9/22 (2006.0 1) House, 10 Salisbury Square, London EC4Y 8JD (GB). (21) International Application Number: (81) Designated States (unless otherwise indicated, for every PCT/GB2010/001384 kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (22) International Filing Date: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 2 1 July 2010 (21 .07.2010) DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (25) Filing Language: English HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (26) Publication Language: English ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (30) Priority Data: NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, 0912637.6 2 1 July 2009 (21 .07.2009) GB SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, 61/235,1 77 19 August 2009 (19.08.2009) US TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (63) Related by continuation (CON) or continuation-in-part (84) Designated States (unless otherwise indicated, for every (CIP) to earlier application: kind of regional protection available): ARIPO (BW, GH, US 61/235177 (CIP) GM, KE, LR, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, Filed on 19 August 2009 (19.08.2009) ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, (71) Applicant (for all designated States except US): EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, BIOTEC PHARMACON ASA [NO/NO]; Strandgata 3, LV, MC, MK, MT, NL, NO, PL, PT, RO, SE, SI, SK, N-9008 Tromsø (NO). SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (71) Applicant (for BB only): GARDNER, Rebecca [GB/GB]; Dehn, St Bride's House, 10 Salisbury Square, Published: London EC4Y 8JD (GB). — with international search report (Art. 21(3)) (72) Inventors; and — before the expiration of the time limit for amending the (75) (for US only): Inventors/Applicants ELDE, Morten claims and to be republished in the event of receipt of [NO/NO]; Stallovegen 104, N-901 6 Tromso (NO). amendments (Rule 48.2(h)) LANES, Olav [NO/NO]; Kveldrovegen 12, N-9007 Tromso (NO). GJELLESVIK, Dag, Rune [NO/NO]; — with sequence listing part of description (Rule 5.2(a)) Sommerlystvegen 27B, N-9012 Tromso (NO). (54) Title: A METHOD OF REMOVING NUCLEIC ACID CONTAMINATION IN REVERSE TRANSCRIPTION AND AM- PLIFICATION REACTIONS (57) Abstract: The invention provides methods of removing nucleic acid contamination from reverse transcription reactions and hot-start PCR, wherein said hot-start PCR is a barrier hot-start PCR set up and/or involves a hot-start DNA polymerase, which methods comprise use of a DNase that is substantially irreversibly inactivated by heating at a temperature of about 5O0C for 5 minutes, and that is substantially specific for double stranded DNA. The invention further provides a DNase that is substantially irreversibly inactivated by heating at a temperature of about 5O0C for 5 minutes, and that is substantially specific for double stranded DNA, nucleic acids encoding said DNase and kits or compositions comprising said DNase or said nucleic acid. A method of removing nucleic acid contamination in reverse transcription and amplification reactions The present invention relates to the removal of contaminating DNA from reverse transcription reaction mixtures, hot-start DNA polymerase preparations and hot-start PCR reaction mixtures through the use of a DNase. The invention also relates to the prevention of false positive results in nucleic acid amplification reactions through the use of a DNase, in particular amplification reactions which involve reverse transcription of the target sequence, a hot-start DNA polymerase and/or a barrier hot-start PCR set-up. The invention also relates to an extremely thermolabile DNase suitable for use in such methods. Nucleic acid amplification techniques such as polymerase chain reactions (PCR's) are one of the most powerful tools available in biotechnology, allowing preparation of a large number of copies of a target sequence from a sample containing only a small amount of nucleic acid. In the case of PCR, oligonucleotide primers complementary to their respective strands of a double stranded target sequence are added to the reaction mixture containing the target sequence and free nucleotides. Thermal cycling in the presence of a DNA polymerase results in amplification of the sequence between the primers. The ability of the amplified fragments created by the PCR process to act as templates for subsequent PCR cycles results in the rapid production of a considerable quantity of the target sequence. Even a single copy of the target sequence can yield sufficient nucleic acid to allow detection by, e.g. hybridization with a labelled probe or incorporation of a 32P labelled deoxynucleotide triphosphates into the amplified segment. Ligase amplification reaction (LAR) also known as ligase chain reaction (LCR), like PCR, uses repetitive cycles and alternating temperature to achieve an exponential increase in the number of copies of the target sequence. In this method, DNA ligase catalyses the joining of two oligonucleotides complementary to adjacent regions of one of the target DNA strands. Two other oligonucleotides complementary to the other strand can also be ligated. After denaturation, the original template strands and the two ligated pairs can act as templates for further hybridisation and ligation. Strand displacement amplification (SDA) exploits the property of the enzymes involved in DNA excision DNA repair to replace a single nicked strand of DNA in a DNA duplex with a newly synthesised strand. To create a nicked single strand repeatedly an endonuclease restriction enzyme, e.g. Hindi or BsoBI, is used which only nicks DNA on one strand of its recognition site when the opposite strand is hemiphosphorothiolated. The primers used in this method contain an appropriate recognition site and dATPαS is used in the polymerisation reaction. Nucleic acid sequence based amplification (NASBA) also known as 3SR (SeIf- Sustaining Sequence Replication) is essentially an in vitro version of natural retroviral transcription. 3SR involves repetitive reverse transcription from the RNA template to form a cDNA template. From the cDNA template an RNA polymerase produces the corresponding RNA. Loop-mediated isothermal amplification (LAMP; Notomi, T., etal, Nuc. Acid Res. 2000 VoI 28 (12) e63) is based on the principle of autocycling strand displacement DNA synthesis. A DNA polymerase with a high strand displacement activity is used (e.g. Bst DNA polymerase large fragment) with specifically designed primers. This process involves strand separation to reveal new target sequence without the need for strand melting (the process is therefore isothermal). Reverse transcription is a process in which a single strand RNA (ssRNA) template is transcribed into a complementary single stranded DNA. The single stranded DNA may then be used to form double strand DNA (dsDNA). Some enzymes are capable of producing the first DNA strand and synthesising the second strand to form dsDNA and others are specific for just one of the two steps. The ssDNA and dsDNA may then be used in a variety of molecular biology applications. For instance they could be used directly in probe based detection assays (e.g. Southern bfotting), sequencing experiments or in cfoning protocofs. Very often the cDNA will be further amplified in an amplification reaction such as PCR, LCR, SDA, LAMP or 3SR for example to provide more material for the above experiments or to be able to quantify the amount of RNA template present in the original sample. Reverse transcription linked amplification reactions can be "one step" or "two step" processes. In a one step process the components of the reverse transcription reaction and the nucleic acid amplification reaction are present in a single reaction vessel and typically the early reaction conditions are selected to allow the reverse transcription reaction to proceed to completion and reaction conditions are then switched to conditions suitable to allow the nucleic acid amplification reaction to proceed. In a two step process the components of the reverse transcription reaction are first combined and the reverse transcription reaction is performed. The reverse transcription product is then combined with the components of the amplification reaction and subjected to the amplification reaction. In a "one tube" two step protocol the amplification reaction components are added to the same reaction vessel in which the reverse transcription reaction was performed. In a "two tube" two step protocol the amplification reaction is performed in a fresh reaction vessel. Reverse transcription can be combined with any of PCA, LAMP, LCR, SDA or BSR in a one or two step process. In the case of SDA a thermostable strand displacing enzyme and a thermostable restriction enzyme (e.g. BsoB1) should be chosen. The ability of these amplification techniques to amplify minute quantities of a target sequence makes them highly susceptible to contamination by genomic DNA in the case of RNA target sequences (i.e. those amplification reactions following or using reverse transcription), and by target sequences in DNA molecules from previous amplification reactions, both of which may be carried over in reagents (e.g. the polymerase, the primers, the reaction buffer, etc.), pipetting devices, laboratory surfaces, gloves or aerosolization.
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