METHODS OF IDENTIFYING ENZYMES AND MICROORGANISMS

Abstract

The invention provides a method of identifying a microorganism that expresses a nucleic acid-modifying enzyme, in sample, the method comprising: (a) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample; (b) adding a further nucleic acid molecule to the sample which nucleic acid molecule is ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme to form a ligation product which comprises a linear single strand of nucleic acid that is capable of being detected; and (c) detecting the presence of the ligation product.

Claims

1. A method of identifying a microorganism that expresses a nucleic acid-modifying enzyme, in a sample, the method comprising: (a) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample; (b) adding a further nucleic acid molecule to the sample which nucleic acid molecule is ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme to form a ligation product which comprises a linear single strand of nucleic acid that is capable of being detected; and (c) detecting the presence of the ligation product.

2. A method according to claim 1, wherein step (a) comprises (i) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample, wherein the nucleic acid substrate is immobilised on a surface; and (ii) washing the nucleic acid substrate immobilised on the surface so as to remove substantially all non-specifically bound material from the substrate.

3. A method according to claim 1 or 2, wherein the nucleic acid-modifying enzyme is a DNA modifying enzyme.

4. A method according to any of claims 1-3, wherein the nucleic acid-modifying enzyme is a ligase such as a topoisomerase (e.g. a type I topoisomerase), an integrase, a recombinase or a transposase.

5. A method according to any of claims 1-4, wherein the microorganism is a pathogenic microorganism or a parasitic microorganism.

6. A method according to any of claims 1-5, wherein the microorganism is selected from the group consisting of a virus, a bacteria, a protozoa, a fungus, a mould, an amoeba or a parasitic worm.

7. A method according to any of claims 1-6, wherein the microorganism belongs to the Mycobacteriaceae family, such as one selected from the Mycobacterium genus, optionally wherein the microorganism is Mycobacterium tuberculosis, Mycobacterium bovis, or Mycobacterium smegmatis.

8. A method according to any of claims 1-5, wherein the microorganism belongs to the Plasmodiidae family, such as one selected from the Plasmodium genus, optionally wherein the microorganism is Plasmodium falciparum.

9. A method according to any of claims 1-5, wherein the microorganism is a Streptococcus such as Group B Streptococcus.

10. A method according to any of claims 1-5, wherein the microorganism is a virus such as a retrovirus, optionally wherein the retrovirus is any of Human Immunodeficiency Virus (HIV), simian immunodeficiency virus (SIV), murine leukaemia virus (MLV) or felineimmunodeficiency virus (FIV).

11. A method according to any of claims 1-10, wherein the sample is food or water.

12. A method, according to any of claims 1-10, wherein the sample is from a mammal.

13. A method according to claim 12, wherein the sample is from a human, a cow, a ferret, a badger, a koala, a chicken, a turkey, a duck, a rodent, an elephant, a bird, a pig, a deer, a coyote, a camel, a puma, a fish, a dog, a sheep, a goat, a cat, or a non-human primate.

14. A method according to claim 12 or 13, wherein the sample is blood plasma, blood serum, whole blood, sputum, saliva, urine, a cell smear, faeces, cerebrospinal fluid or a biopsy.

15. A method according to any of claims 1-14 wherein step (a) comprises incubating the nucleic acid substrate and sample at a temperature of between 30-40 C., preferably 37 C.

16. A method according to any of claims 1-15, wherein the nucleic acid substrate is selectively targeted by the nucleic acid-modifying enzyme from the microorganism.

17. A method according to any of claims 1-16, wherein the nucleic acid substrate is single stranded or double stranded.

18. A method according to any of claims 1-17, wherein the microorganism is selected from the Plasmodium genus, and the nucleic acid substrate is double stranded DNA wherein at least one strand comprises a sequence selected from the group consisting of ACTACCATTCTGAGTCGTTCGAAGTTCCTATACTTT (SEQ ID No: 1) and TCTAGAAAGTATAGGAACTTCGAACGACTCAGAATG (SEQ ID No: 2); a sequence sharing at least 90% sequence identity thereto; or a sequence which is a part of at least 5 consecutive nucleotides of any of said sequences.

19. A method according to any of claims 1-17, wherein the microorganism is selected from the Plasmodium genus, and the nucleic acid substrate is double stranded DNA wherein at least one strand comprises a sequence selected from the group consisting of ACTACCATTCTGAGTCGTTCGATCTAAAAGACTTAGA (SEQ ID No: 3) and ATTTTTCTAAGTCTTTTAGATCGAACGACTCAGAATG (SEQ ID No: 4); a sequence sharing at least 90% sequence identity thereto; or a sequence which is a part of at least 5 consecutive nucleotides of any of said sequences.

20. A method according to any of claims 1-17, wherein the microorganism is selected from the Mycobacterium genus, and the nucleic acid substrate is single stranded DNA that comprises the sequence CAGTGAGCGAGCTTCCGCTTGACATCCCAATAGTTTCTCTTC (SEQ ID No: 5); a sequence sharing at least 90% sequence identity thereto; or a sequence which is a part of at least 5 consecutive nucleotides of any of said sequence.

21. A method according to any of claims 1-17, wherein the microorganism is a virus, and the nucleic acid substrate is a long terminal repeat (LTR) sequence that is selectively targeted by an integrase of the virus.

22. A method according to claim 21, wherein the nucleic acid substrate is double stranded DNA wherein at least one strand comprises a sequence selected from the group consisting of TTTAGTCAGTGTGGAAAATCTCTAGCAGT (SEQ ID No: 6) and ACTGCTAGAGATTTTCCACACTGACTAAA (SEQ ID No: 7); a sequence sharing at least 90% sequence identity thereto; or a sequence which is a part of at least 5 consecutive nucleotides of any of said sequences; or wherein the nucleic acid substrate is double stranded DNA wherein at least one strand comprises a sequence selected from the group consisting of TTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID No: 22) and AATGAAAGACCCCCGCTGACGGGTAGTCAA (SEQ ID No: 23); a sequence sharing at least 90% sequence identity thereto; or a sequence which is part of at least 5 consecutive nucleotides of any of said sequences.

23. A method according to claim 22, wherein the nucleic acid substrate is double stranded DNA wherein the first strand comprises the sequence TTTAGTCAGTGTGGAAAATCTCTAGCAGT (SEQ ID No: 6) and the second strand comprises the sequence ACTGCTAGAGATTTTCCACACTGACTAAA (SEQ ID No: 7), or wherein the nucleic acid substrate is double stranded DNA wherein the first strand comprises the sequence TTGACTACCCGTCAGCGGGGGTCTTTCATT (SEQ ID No: 22) and the second strand comprises the sequence AATGAAAGACCCCCGCTGACGGGTAGTCAA (SEQ ID No: 23).

24. A method according to any of claims 1-23, wherein the surface is a solid support.

25. A method according to claim 24, wherein the solid support is glass, paper, cardboard, sepharose, agarose, plastic, metal, silicon, ceramics and latex.

26. A method according to claim 25, wherein the solid support is coated with maleic anhydride.

27. A method according to any of claims 1-26, wherein ligation of the further nucleic acid molecule to the nucleic acid substrate does not generate a circular ligation product.

28. A method according to any of claims 1-27, wherein the ligation product is formed directly following ligation of the further nucleic acid molecule to the nucleic acid substrate.

29. A method according to any of claims 1-27, wherein the ligation product is formed indirectly via the formation of one or more intermediate products following ligation of the further nucleic acid molecule to the nucleic acid substrate.

30. A method according to any of claims 1-29, wherein the further nucleic acid molecule is double stranded DNA or single stranded DNA.

31. A method according to claim 30, wherein the further nucleic acid molecule is double stranded DNA, the nucleic acid substrate is a long terminal repeat (LTR) sequence that is selectively targeted by an integrase of the virus, and the nucleic acid-modifying enzyme is an integrase.

32. A method according to any of claims 1-31, wherein the ligation product is one that comprises a linear single strand of nucleic acid that is capable of being detected by hybridising to one or more detectable oligonucleotides, or that is capable of being detected by comprising one or more detectable nucleotides.

33. A method according to claim 32, wherein the one or more detectable oligonucleotides are capable of forming, by self-assembly, with other such one or more detectable oligonucleotides, substantially contiguous single strands of a double-stranded nucleic acid filament.

34. A method according to claim 32 or 33, wherein the one or more detectable oligonucleotides or nucleotides are detectably labelled with one or more fluorescent dyes, radioactive nucleotides or biotinylated nucleotides, or wherein the one or more detectable oligonucleotides or nucleotides are detectably labelled by being coupled to an enzyme, which enzyme is capable of converting a substrate into a detectable product.

35. A method according to claim 34, wherein the one or more detectable oligonucleotides comprise biotinylated nucleotides which are detected by using streptavidin coupled gold nanoparticles.

36. A method according to any of claims 32-35, wherein the one or more detectable oligonucleotides comprise a sequence selected from the group consisting of CAG TGA GCG TCT GGC TGA AGC TTC CGC T (SEQ ID No: 16) and CCA GAC GCT CAC TGA GCG GAA GCT TCA G (SEQ ID No: 17).

37. A method according to any of claims 30-36, wherein the further nucleic acid molecule is double stranded DNA wherein the first strand comprises the sequence TGCACGCATGTCGATGTGTCGCAATCGCATGTTGTCATCGTGCATGCATCATGACG TGCTGACTGAAGCTTCCGCT (SEQ ID No: 12) and the second strand comprises the sequence TCAGCACGTCATGATGCATGCACGATGACAACATGCGATTGCGACACATCGACATG CGTGCAAGCGGAAGCTTCAG (SEQ ID No: 13).

38. A method according to any of claims 30-36, wherein the further nucleic acid molecule is single stranded DNA, the nucleic acid substrate is single or double stranded DNA that is selectively targeted by a type I topoisomerase, and the nucleic acid-modifying enzyme is a type I topoisomerase.

39. A method according to claim 38, wherein the further nucleic acid molecule comprises the sequence CAGTGAGCGTCTGGCTGAAGCTTCCGCT (SEQ ID No: 14) or TTCTAGACCAGACGCTCACTGAGCGGAAGCTTCAG (SEQ ID No: 15).

40. A method according to any of claims 1-39, wherein step (c) is followed by a wash step to remove substantially all further nucleic acid molecule that is non-ligated to the substrate.

41. A method of identifying a microorganism that expresses a nucleic acid-modifying enzyme, in a sample, the method comprising: (a) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample, wherein the nucleic acid substrate is capable of being processed in the presence of the nucleic acid modifying enzyme to form a ligation product which comprises a linear single strand of nucleic acid that is capable of being detected; and (b) detecting the presence of the ligation product.

42. A method of identifying a microorganism that expresses a nucleic acid-modifying enzyme, in a sample, the method comprising: (a) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample, wherein the nucleic acid substrate comprises a first strand of nucleic acid that is immobilised on a surface and a second strand of nucleic acid that is hybridised to the first strand of nucleic acid but which is not immobilised to the surface, and wherein, in the presence of the nucleic acid modifying enzyme, the first strand of nucleic acid is capable of being ligated to the second strand of nucleic acid to form a ligation product which comprises a linear single strand of nucleic acid that is capable of being detected, (b) washing the nucleic acid substrate immobilised on the surface so as to remove substantially all non-specifically bound material from the substrate, under denaturing conditions; and (c) detecting the presence of the ligation product.

43. A method according to claim 42, wherein the surface is a bead or a slide.

44. A method according to claim 43, wherein the bead is a streptavidin coated bead and the first strand of the nucleic acid substrate is a biotinylated strand of nucleic acid.

45. A method according to claim 44, wherein the slide is a codelink slide and the first strand of the nucleic acid substrate is an amine-linked strand of nucleic acid.

46. A method according to any of claims 1-45, wherein the presence of the ligation product is detected by any one or more techniques selected from the group consisting of a linear amplification technique, southern blotting, polymerase chain reaction (PCR), reverse-transcription-PCR (RT-PCT), quantitative PCR (qPCR), restriction fragment length dimorphism-PCR (RFLD-PCR), primer extension, DNA array technology, and isothermal amplification.

47. A method according to any of claims 1-45, wherein the presence of the ligation product is detected by an amplification technique that does not involve use of a polymerase.

48. A method according to claim 47, wherein the amplification technique involves the use of one or more detectable oligonucleotides as defined in any of claims 32-36.

49. A method according to any of claims 1-48, wherein the presence of the ligation product is detected in a microfluidic system.

50. A method according to claim 49, comprising: (i) loading the washed nucleic acid substrate from step (b) and the further nucleic acid molecule from step (c) into a sample chamber comprising a flow through channel, wherein droplets comprising the nucleic acid substrate are generated; (ii) transferring the droplets from the sample chamber to a droplet retaining means through the flow through channel; (iii) capturing one or more single droplets in individual cavities of the droplet retaining means, wherein each single droplet is spatially isolated from other droplets; and (iv) detecting, in one or more captured droplets, the ligation product.

51. A method according to claim 50, wherein the sample chamber comprises one or more inlet channels and/or one of more outlet channels for the generated drops.

52. A method according to claim 50 or 51, wherein the one or more flow through channels, inlet channels and/or outlet channels have a diameter of 10-50 micrometers, such as approximately 25 micrometers.

53. A method of diagnosing an infectious disease in a subject, the method comprising identifying a microorganism in a sample from the subject according to the method of any of claims 1-52, wherein the presence of the microorganism in the sample is indicative of the infectious disease.

54. A method according to claim 53, wherein the disease is tuberculosis; or malaria; or Group B Streptococcus disease; or a retroviral infection such as HIV infection or FIV infection or MLV infection or SIV infection; or cancer.

55. A method according to claim 53 or 54, wherein the subject is a mammal, a human, a cow, a ferret, a badger, a koala, a chicken, a turkey, a duck, a rodent, an elephant, a bird, a pig, a deer, a coyote, a camel, a puma, a fish, a dog, a sheep, a goat, a cat, or a non-human primate.

56. A method of combating an infectious disease in a subject, the method comprising diagnosing the disease according to the methods of any of claims 53-55, and treating the disease.

57. An anti-infectious disease agent for use in combating an infectious disease in a subject who has been diagnosed with the disease according to the methods of any of claims 53-55.

58. A method of assessing whether an agent has an effect on an infectious disease caused by a microorganism in a subject, the method comprising administering the agent to the subject and determining the effect of the agent on the amount of ligation product produced in a sample from the subject when the sample is contacted with a nucleic acid substrate targeted by a nucleic acid-modifying enzyme of the microorganism and a further nucleic acid molecule that is ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme, according to the method of any of claims 1-52.

59. A method of assessing whether an agent has an effect on a microorganism in a sample, the method comprising: (a) contacting the sample with the agent; and (b) assessing the effect of the agent on the amount of ligation product produced when the sample is contacted with a nucleic acid substrate targeted by a nucleic acid-modifying enzyme of the microorganism and a further nucleic acid molecule that is ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme, according to the method of any of claims 1-53.

60. A method of assessing the presence or activity of a nucleic acid-modifying enzyme in a sample, the method comprising: (a) contacting a nucleic acid substrate targeted by the nucleic acid-modifying enzyme with the sample, optionally wherein the nucleic acid substrate is immobilised on a surface, and the nucleic acid substrate immobilised on the surface is washed so as to remove substantially all non-specifically bound material from the substrate; (b) adding a further nucleic acid molecule to the sample which nucleic acid molecule is ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme to form a ligation product that comprises a linear single strand of nucleic acid that is capable of being detected; and (c) detecting the presence or activity of the ligation product.

61. A device for carrying out a method according to any of claims 1-52 and 60.

62. A device according to claim 61, wherein the device comprises a means for washing the nucleic acid substrate immobilised on the surface according to step (a)(i) of claim 1.

63. A device according to claim 61 or 62, wherein the device comprises a separate reaction chamber and detection chamber.

64. A device according to any of claims 61-63, wherein the device comprises a control chamber.

65. A device according to any of claims 61-64, wherein the device comprises a waste outlet.

66. A device according to any of claims 61-65, wherein the device comprises a means that allows the ligation product to be detected by an amplification technique according to claim 47 or 48.

67. A device according to any of claims 61-66, further comprising one or more analytical means to detect the ligation product.

68. A kit of parts comprising a nucleic acid substrate targeted by a nucleic acid-modifying enzyme and a means for detecting a ligation product formed by ligation of the nucleic acid substrate to a further nucleic acid molecule, which ligation product comprises a linear single strand of nucleic acid that is capable of being detected.

69. A kit of parts according to claim 68, wherein the nucleic acid substrate is immobilised on a surface, optionally as a surface as defined in any of claims 24-26.

70. A kit of parts according to claim 68 or 69, wherein the means for detecting the ligation product comprises one or more detectable oligonucleotides as defined in any of claims 33-36.

71. A kit of parts according to any of claims 68-70, wherein the nucleic acid substrate is as defined in any of claims 16-23.

72. A kit of parts according to any of claims 68-71, wherein the kit further comprises a further nucleic acid molecule that is capable of being ligated to the nucleic acid substrate in the presence of the nucleic acid-modifying enzyme to form a ligation product which comprises a linear single strand of nucleic acid that is capable of being detected.

73. A kit of parts according to claim 72, wherein the further nucleic acid molecule is as defined in any of claims 30, 31 and 37-39.

74. A kit of parts according to any of claims 68-73, which kit does not comprise a polymerase.

75. A kit of parts according to any of claims 68-74, wherein the kit comprises a plurality of, nucleic acid substrates each targeted by a nucleic acid-modifying enzyme of a different microorganism, cell or cell type.

76. A kit of parts according to any of claims 68-75, wherein the kit comprises an agent for depletion of divalent cations.

Description

[0277] The invention will now be described with the aid of the following Figures and Examples.

[0278] FIG. 1: HIV integrase detection by SHOfLE methodology (see Example 3). SHOfLE was performed as described in the text using purified HIV integrase. The integrase was diluted in storage buffer (200 mM KCl, 10 M ZnCl.sub.2, 50% glycerol) as indicated on the figure. The no integrase control was just added storage buffer. The reaction was carried out in retrovirus reaction buffer supplemented with a double stranded DNA substrate (the further nucleic acid) and 10% (v/v) purified undiluted or diluted integrase.

[0279] FIG. 2: Comparison of the SHOfLE strategy to current diagnostic methods

[0280] FIG. 3: Comparison of the microscopy based readout to the strip well based read out.

[0281] FIG. 4: Detection of the malaria parasite (via pfTopol) in blood samples from malaria infected patients. The blood samples had only been frozen once at the time of analysis. The blood samples were lysed as described and subjected to SHOfLE. The infected samples were compared to blood samples from seven uninfected individuals. One of the SHOfLE oligonucleotides was labeled with Fam and the products were hence visible by fluorescence microscopy.

[0282] FIG. 5: Detection of the malaria parasite (via pfTopol) in blood samples from malaria infected patients. The blood samples had only been frozen once at the time of analysis. The blood samples were lysed as described and subjected to SHOfLE. The infected samples were compared to blood samples from four uninfected individuals. One of the SHOfLE oligonucleotides was labeled with biotin and the products was visible by binding to streptavidin conjugated gold nanoparticles and subsequent silver precipitation on the gold nanoparticles.

[0283] FIG. 6: Detection of the malaria parasite (via pfTopol) in blood samples from 30 malaria infected patients. The blood samples had been frozen twice at the time of analysis. The blood samples were lysed as described and subjected to SHOfLE. The infected samples were compared to blood samples from 18 uninfected individuals. One of the SHOfLE oligonucleotides was labeled with biotin and the products was visible by binding to streptavidin conjugated gold nanoparticles and subsequent silver precipitation on the gold nanoparticles.

[0284] FIG. 7: Graph showing quantification of the data in FIG. 6.

[0285] FIG. 8: Summary of conclusions.

[0286] FIG. 9: Cultured parasites was lysed by mixing 80 L of blood with 320 L TE (10 mM Tris-HCl, pH 7,5 and 1 mM EDTA). This mixture was subjected to bead beating for 1, 2, or 3 minutes. The lysate was analyzed by SHOfLE as described for vortexed samples.

[0287] FIG. 10: SHOfLE detection (silver stain) of different amounts of purified pfTopol.

[0288] FIG. 11: REEAD detection (incorporation of biotin during RCA, silverstain as for SHOfLE) of different amounts of purified pfTopol.

[0289] FIG. 12: Summary of conclusions.

[0290] FIG. 13: Comparison of SHOfLE and REEAD assays.

[0291] FIG. 14: Schematic depiction of the SHOfLE strategy.

[0292] FIG. 15: Schematic depiction of retrovirus detection by SHOfLE

[0293] FIG. 16: Schematic depiction of msTopol (or other mycobacterial type 1A topoisomerase) SHOfLE.

[0294] FIG. 17: Schematic depiction of detection of nucleic acid-modifying enzyme where ligation product is formed indirectly and further nucleic acid molecule is part of nucleic acid substrate. (A) The nucleic acid substrate comprises two strands, a first strand immobilised to a bead and a second strand hybridised to the first strand but not immobilised to a bead. This second strand corresponds to the further nucleic acid molecule which also comprises a double stranded nucleic acid portion separate from the portion of the further nucleic acid molecule that is hybridised to the first strand of the nucleic acid substrate. The double stranded nucleic acid portion of the further nucleic acid molecule may comprise one or more detectable oligonucleotides, denoted in the figure as stars. In the presence of a nucleic acid-modifying enzyme (target), the first and second strands of the nucleic acid substrate are ligated. The nucleic acid substrate is then washed under denaturing conditions. If the two strands of the nucleic acid substrate have been ligated, then exposure to denaturing conditions results in the formation of a ligation product that comprises a linear single strand of nucleic acid that is capable of being detected. If, as shown in the figure, this linear single strand of nucleic acid comprises one or more detectable oligonucleotides, then the presence of a signal following the wash step is a direct read-out of the present of nucleic acid-modifying enzyme. If the two strands of the nucleic acid substrate have not been ligated, then exposure to denaturing conditions does not result in formation of the ligation product and no signal is detected because the further nucleic acid molecule is separated from the first strand of nucleic acid substrate that remains immobilised on the surface. (B) A more specific embodiment than that shown in (A) where the double stranded nucleic acid portion of the further nucleic acid molecule comprises one or more fluorescently labelled oligonucleotides.

[0295] FIG. 18: PFTopol mediated ligation of a PCT product to a surface. Ligation product comprising linear single strand of nucleic acid is formed either directly (A) or indirectly (B). In (B), the nucleic acid substrate comprises two strands as it does in FIG. 17, and the further nucleic acid molecule is part of this nucleic acid substrate. A first strand of the nucleic acid substrate is immobilised to a codelink slide and a second strand is hybridised to the first strand but is not immobilised to the codelink slide.

[0296] FIG. 19: Detection of pfTopo I in blood lysate.

EXAMPLE 1

Use of SHOfLE Technology to Detect Plasmodium falciparum Topoisomerase I

[0297] Methodology

[0298] Cloning, Expression, and Purification of pfTopo1

[0299] The gene encoding plasmodium falciparum topoisomerase I (pfTopol) was cloned into the pYES2.1 vector from Invitrogen using the pYES2.1 TOPO TA Expression Kit. The resulting vector enables galactose inducible expression of pfTopol in yeast. Purification of pfTopo1 was done essentially as described previously (Lisby et al, J Biol Chem. 2001 June 8; 276(23):20220-7). Purified enzyme was stored at 20 C.

[0300] Blood Sample Preparation

[0301] 8 L of fresh blood sample or a blood sample only frozen once is mixed with 32 L of hypotonic lysis buffer (10 mM Tris-HCL pH 7.5; 5 mM EDTA, 0.2% Tween 20; 1 mM DTT, 1 mM PMSF) and 100 L of Pico-Surf (TM) 1 (2% in Novec 7500). Droplets are generated by vortexing the samples for one minute. The droplets are incubated on ice for 15 minutes and broken by addition of 15 L 1H,1H,2H,2H-Perfluoro-1-octanol.

[0302] Coupling of Amine Labelled Substrate to Microtiter Wells

[0303] The substrate for pfTopol is a DNA duplex consisting of two amine coupled 36-meric oligonucleotides. To immobilize the substrate on Well-Coated Amine Binding, 8-well strip plates from G-biosciences, we first couple one strand covalently to the wells and then hybridize the other strand to the covalently attached oligonucleotide.

[0304] Covalent Coupling of Amine Conjugated Oligonucleotide to Microtiter Wells

[0305] The amine-couple oligonucleotide ([AmC6T]actaccattctgagtcgttcgaagttcctatacttt; SEQ ID No: 1) is diluted in 50 mM sodium-phosphate buffer, pH 8.5 to a final concentration of 5 M. 50 L of this solution is added to each amine binding well.

[0306] The wells are incubated over night at room temperature in a humidity chamber containing a saturated NaCl solution.

[0307] Next day, the wells are processed as follows: 30 minutes in warm (50 C.) blocking buffer, 21 minute wash in water, 30 minutes in warm (50 C.) wash buffer 1, 21 minute wash in water. Let the wells air-dry.

[0308] Hybridization of Second Substrate Strand to the Covalently Attached Strand

[0309] The second substrate strand ([AmC6T]TCTAGAAAGTATAGGAACTTCGAACGACTCAGAATG; SEQ ID No: 2) is diluted to a final concentration of 0.2 M in a buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, and 300 mM NaCl. 50 L of this solution is added to each well. Incubate at 37 C. for 1 h in humidity chamber. Wash for 1 min in wash buffer 2, 1 min in wash buffer 3, and 1 min in 96% ethanol. Let the wells air-dry.

[0310] Topoisomerase I Reaction

[0311] Prepare a solution containing 20% (v/v) broken droplets (see Blood sample preparation) or 10% (v/v) purified pfTopol (different concentrations as indicated on figure) as well as 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 300 mM NaCl. Add 50 L to the each microtiter well. Incubate for 1 h at 37 C. in humidity chamber. Wash 35 minutes in wash buffer 4. To each well, add 50 L of Ligation mixture: 2 M linker oligo (TTCTAGACCAGACGCTCACTGAGCGGAAGCTTCAG; SEQ ID No: 15); 300 mM NaCl; 1 mM Tris-HCl (pH 7.5); 5 mM MgCl.sub.2. Incubate at 37 C. for 1 h in humidity chamber. Wash for 1 min in wash buffer 2, 1 min in wash buffer 3, and 1 min in 96% ethanol. Let the wells air-dry.

[0312] SHOfLE

[0313] Add 50 L of SHOfLE mixture to each well: 2SSC, 20% formamid, 5% glycerol, and 0.2 M of each of the biotin labelled SHOfLE oligoes ([Btn]CAG TGA GCG TCT GGC TGA AGC TTC CGC T; SEQ ID No: 16; [Btn]CCA GAC GCT CAC TGA GCG GAA GCT TCA G; SEQ ID No: 17). Incubate at 50 C. for 3 h in humidity chamber. Wash for 20 min in wash buffer 2, 10 min in wash buffer 3 and 1 min in 96% ethanol. Let the wells air-dry.

[0314] Silver Staining

[0315] Strep-Gold reaction mix: 2% (v/v) streptavidin coupled gold nanoparticles (Nanocs; cat. no. GNA3; 0.05% w/v), 0.5% BSA in 0.1 M PBS, pH 10+10 l of 5% BSA. Add 50 L to each well and incubate at room temperature for 15 minutes. Wash for 5 minutes in wash buffer 2, wash for 5 minutes in wash buffer 3, rinse in water, and wash for 1 min in 96% ethanol. Let the wells air-dry.

[0316] Solution A and Solution B from the silver enhancer kit (SigmaAldrich, SE100) are mixed 1:1 immediately before use and applied to the wells. Develop for 10 minutes. After development, the wells are rinsed in water and dried. The amount of silver precipitated is quantified using a POLARstar Omega fluorescence reader.

[0317] Buffer

[0318] Blocking buffer: 50 mM Ethanolamine, 0.1 M Tris (pH 9.0)

[0319] Wash buffer 1: 4SSC; 0.1% SDS

[0320] Wash buffer 2: 0.1M Tris-HCl (pH 7.5); 150 mM NaCl; 0.3% SDS

[0321] Wash buffer 3: 0.1M Tris-HCl (pH 7.5); 150 mM NaCl; 0.05% Tween 20

[0322] Wash buffer 4: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1M NaCl

EXAMPLE 2

Use of SHOfLE Technology to Detect Mycobacterial Topoisomerase I

[0323] Summary

[0324] As a model system for mycobacterial topoisomerase I, we use topoisomerase I from Mycobacterium smegmatis (msTopol). To enable SHOfLE detection of msTopol, we immobilize a single stranded substrate for msTopol. msTopol can mediate ligation of one of the SHOfLE oligonucleotides to this substrate strand and thereby create a base for SHOfLE hybridization.

[0325] Methodology

[0326] Coupling of msTopol Substrate to an Amine Binding Surface (CodeLink Slide)

[0327] The msTopol substrate 5-CAG TGA GCG AGC TTC CGC TTG ACA TCC CAA TAG TTT CTC TTC-Amino-C6-3 (SEQ ID No: 5) is coupled to CodeLink slides (SurModics) as described below: The amine-couple oligonucleotide is diluted in 50 mM sodium-phosphate buffer, pH 8.5 to a final concentration of 5 M. 1 L of this solution is added to a small area of a CodeLink slide (marked using a hydrophobic PAP pen). The oligonucleotide conjugated area of the CodeLink slide is denominated the printed area.

[0328] The slide is incubated over night at room temperature in a humidity chamber with saturated salt.

[0329] Next day, the slide is processed as follows: 30 minutes in warm (50 C.) blocking buffer, 21 minute wash in water, 30 minutes in warm (50 C.) wash buffer 1, 21 minute wash in water. Let the slide air-dry.

[0330] msTopo Binding Reaction

[0331] Prepare a solution containing msTopol as well as 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, and 300 mM NaCl. Add 5 L to each printed area (see previous section). Incubate for 1 h at 37 C. in humidity chamber. Wash 35 minutes in wash buffer 4. To each printed are, add 5 L of ligation mixture: 2 M oligonucleotide ([Btn]CAG TGA GCG TCT GGC TGA AGC TTC CGC T; SEQ ID No: 16); 300 mM NaCl; 1 mM Tris-HCl (pH 7.5); 5 mM MgCl.sub.2. Incubate at 37 C. for 1 h in humidity chamber. Wash for 1 min in wash buffer 2, 1 min in wash buffer 3, and 1 min in 96% ethanol. Let the slide air-dry.

[0332] SHOfLE

[0333] As described for malaria SHOfLE, except the biotins on the SHOfLE oligonucleotide is replaced by a fluorophore (FAM). (See Example 1.)

[0334] Microscopy

[0335] After SHOfLE hybridization, the slide is washed for 20 min in wash buffer 2, 10 min in wash buffer 3, 1 min in 96% ethanol, and air-dried. The slide is mounted using Vectashield and analyzed by fluorescence microscopy. 12 random pictures were acquired for each experimental condition and the fluorescence intensity was quantified using Image J software.

[0336] Buffers

[0337] As for malaria SHOfLE (see Example 1).

EXAMPLE 3

Use of SHOfLE Technology to Detect HIV Integrase

[0338] Summary

[0339] To enable SHOfLE detection of HIV integrase we immobilize the LTR (the sequence binding the HIV integrase) and allow the integrase to ligate the LTR to a double stranded DNA substrate. The ends of the double stranded DNA is designed in such a way, that the SHOfLE oligonucleotides will hybridize to them allowing detection of integration events by SHOfLE.

[0340] Cloning, Expression, and Purification of HIV Integrase

[0341] The gene encoding the HIV integrase was purchased from Geneart (Life Technologies) and cloned into the pTrcHis vector from Invitrogen using the pTrcHis TOPO TA Expression Kit. The resulting vector enables IPTG inducible expression of His-tagged integrase in E. coli. Transformed BL21 cells were grown in LB and expression of the integrase was induced with IPTG (0.3 mM). His-tagged integrase was purified using a Ni-NTA Superflow column and eluted with elution buffer (10 mM Tris-HCl, pH7.5, 200 mM NaCl, 150 mM imidazole, 5 mM MgCl.sub.2, 10% glycerol, 1 mM PMSF). lntegrase containing fractions were pooled and dialyzed against 200 mM KCl, 10 M ZnCl.sub.2, 50% glycerol. Aliquots of fractions were stored at 20 C.

[0342] Preparation of Double Stranded DNA Substrate

[0343] The following two oligonucleotides are hybridized in reaction buffer (20 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.2), 200 mM KCl, 10 mM MnCl.sub.2, 10 mM DTT, and 10% glycerol). The final concentration of each oligonucleotide in the hybridization mixture was 5 M.

TABLE-US-00001 (SEQIDNo:12) 5-tgcacgcatgtcgatgtgtcgcaatcgcatgttgtcatcgtgcatgc atcatgacgtgctgactgaagcttccgct (SEQIDNo:13) 5-tcagcacgtcatgatgcatgcacgatgacaacatgcgattgcgacac atcgacatgcgtgcaagcggaagcttcag

[0344] Coupling of Amine LTR to Microtiter Wells

[0345] The LTR sequence that binds HIV integrase is immobilized on the substrate of Well-Coated Amine Binding, 8-well strip plates from G-biosciences, by coupling one strand covalently to the wells and then hybridizing the other strand to the covalently attached oligonucleotide.

[0346] Covalent Coupling of Amine Conjugated LTR Oligonucleotide to Microtiter Wells

[0347] The amine-couple oligonucleotide (5-[AmC6T] TTTAGTCAGTGTGGAAAATCTCTAGCAGT: SEQ ID No: 6) is coupled to the Well-Coated Amine Binding, 8-well strip plates from G-biosciences as described for malaria SHOfLE (see Example 1).

[0348] Hybridization of Second LTR Strand to the Covalently Attached Strand

[0349] The second LTR strand (5-[AmC6T]ACTGCTAGAGATTTTCCACACTGACTAAA; SEQ ID No: 7) hybridized to the covalently attached strand as described for malaria SHOfLE (see Example 1).

[0350] Integrase Reaction

[0351] Dilute the double stranded DNA substrate in reaction buffer (20 mM 2-(N-morpholino) ethanesulfonic acid (MES, pH 6.2), 200 mM KCl, 10 mM MnCl.sub.2, 10 mM DTT, and 10% glycerol). The final concentration should be 0.5 M. Add either 2% (v/v) of purified enzyme (diluted as indicated on the graph) or HIV-integrase storage buffer (200 mM KCl, 10 M ZnCl.sub.2, 50% glycerol). Add 50 L to each well and incubate at 37 C. for 2 h in a humidity chamber. Wash for 1 min in wash buffer 2, 1 min in wash buffer 3, and 1 min in 96% ethanol. Let the wells air-dry.

[0352] SHOfLE

[0353] As described for malaria SHOfLE (see Example 1).

[0354] Silver Staining

[0355] As described for malaria SHOfLE (see Example 1).

[0356] Buffers

[0357] As for malaria SHOfLE (see Example 1).

EXAMPLE 4

Detection of Plasmodium falciparum Topoisomerase I by One-Step Assay

[0358] Summary

[0359] Plasmodium falciparum topoisomerase I (PFTopo I) is detected by immobilizing amine coupled-oligonucleotide to a surface. PFTopo I can mediate ligation of a PCR product to this oligonucleotide to form a ligation product that comprises a linear single strand of nucleic acid that can be detected.

[0360] The method is illustrated in FIGS. 17 and 18(B) and involves only one incubation step and one wash step (which itself may comprise one or more individual washes in different wash liquids). Advantages of the methodology include the fact that the substrate can be prepared beforehand, thus reducing time to readout; the background is lower (substrate more specific according to Plasmodium specificity); and sensitivity is high (see FIG. 19 which shows a detection level below 5 parasites/l).

[0361] Methodology

[0362] Printing Slides

[0363] Day 1: Prepare slides with 2 codelink pieces on each slide: Cut Codelink slide into 55 mm pieces and glue them to an objective glass. Let it dry for 10-15 min and use the grease pen around the edges. Alternatively, make wells with the fatpen.

[0364] In the middle of the codelink piece/fatpen well, place a drop of 10 L of following coupling mixture:

[0365] Coupling Mixture

TABLE-US-00002 6x print buffer 2 L H.sub.2O 4 L Amine-oligo (Am-S5-Cl-37, 10 M) 6 L Final volume 12 L (10 L pr printed area)

[0366] The amine-oligo comprises the sequence:

TABLE-US-00003 (SEQIDNo:3) ACTACCATTCTGAGTCGTTCGATCTAAAAGACTTAGA.

[0367] The slides are places in a humidity chamber with saturated NaCl at 25 C. o/n.

[0368] Day 2: Place the slides in stands and fill with blocking buffer (only enough to cover the codelink pieces), and block for 30 min at 50 C. Wash twice in ion-exchanged water (21 min). Wash for 30 min at 50 C. in wash buffer 1 (4SSC+0.1% SDS). Wash twice in ion-exchanged H.sub.2O for 1 min (place on shaker). Let the slides air dry.

TABLE-US-00004 FROMNOW,EVERYTHINGSHOULDBEDONEINDIMMED LIGHT.

[0369] Hybridization of PCR Product to Primers

TABLE-US-00005 TEN3 9 L 2.5 or 5 ng PCR product (adjusted to 2.5 or 5 ng/L) 1 L

[0370] The PCR product is produced using the vector pYES2.1 TOPO (Invitrogen) as a template.

[0371] Primers:

TABLE-US-00006 P1: (SEQIDNo:25) ATTTTTCTAAGTCTTTTAGATCGAACGACTCAGAAT GATGCATGTATACTAAACTCACAAATTAGAGC P2: (SEQIDNo:26) TTTTTTTTTTTTTTTTTTTTTTTTTGCTTTCTCATA GCTCACGCTG

[0372] The generated PCR fragment is around 1500 bp and hybridizes to the following oligo:

TABLE-US-00007 (SEQIDNo:3) ACTACCATTCTGAGTCGTTCGATCTAAAAGACTTAGA

[0373] Place 10 L per well.

[0374] Incubate 1 hour at 37 or 50 C. in humidity chamber.

[0375] Wash the slides for 1 min in wash buffer 2 (place on shaker). Wash for 1 min in wash buffer 3 (place on shaker).

[0376] Dehydrate the slides for 1 min in 99% EtOH (place on shaker). Let the slides air dry.

[0377] Cleavage of Substrate:

[0378] TEN3 9 L

[0379] pfTop1/blood lysate 1 L

[0380] Incubate the slides for 60 min at 37 C. in a humidity chamber.

[0381] The nucleic acid substrate that is recognised by topoisomerase in this Example is made up of the amine-linked oligonucleotide and the part of the PCR product that it is hybridised to. The amine-linked oligonucleotide is cleaved by topoisomerase and then ligated to the part of the PCR product that it is hybridised to.

[0382] Wash:

TABLE-US-00008 TE-500 mM NaCl 1 min Wash 2 1 min Wash 3 1 min Water 1 min Urea (8M) 30 min Urea (8M) 30 min Wash 2 1 min Wash 3 1 min 96% EtOH 1 min

[0383] Let airdry.

[0384] Mount each slide with 2 L Vectashield without DAPI. Put on coverglass. Add a small drop of immersion oil on the coverglass and analyse under 63 objective.

[0385] Buffers

[0386] 10*TE:

[0387] 100 mM Tris pH 7.5 (for 100 mL, use 10 mL of 1M)

[0388] 10 mM EDTA (for 5 mL of 200 mM)

[0389] TEN3:

[0390] 1 mL of 3M NaCl

[0391] 1 mL of 10*TE

[0392] 7 mL Water

[0393] Preparation of Blood-Lysate

[0394] 320 L lysis buffer (10 mM Tris HCl, pH 7.5; EDTA; protease inhibitors (complete, Roche))+80 L blood.

[0395] Mix with 100 L glass-beads, bead beat for 3 min.

[0396] Leave on ice for 15 min.

[0397] Centrifuge at 1000 g for 5 min.

[0398] Use instead of pfTopo in One-Step-assay protocol.

[0399] Sequences

TABLE-US-00009 MycobacteriumtuberculosistopoisomeraseIgenesequence: SEQIDNO:18 TTGGCTGACCCGAAAACGAAGGGCCGTGGCAGCGGCGGCAATGGCAGCGGCCGGCGACTGGTCAT CGTCGAGTCGCCCACCAAGGCGCGCAAGCTGGCCTCCTACCTGGGCTCTGGCTACATCGTCGAGT CCTCCCGGGGGCACATCCGTGACTTGCCGCGGGCCGCGTCGGATGTACCCGCAAAGTACAAGTCG CAGCCGTGGGCGCGGCTCGGGGTCAACGTCGACGCCGACTTCGAACCGCTCTACATCATCAGCCC GGAGAAACGGAGCACCGTCAGCGAGCTCAGGGGCCTGCTCAAAGACGTGGACGAGCTGTATCTGG CCACGGATGGGGACCGTGAGGGCGAAGCTATTGCCTGGCATCTGCTGGAAACCCTCAAACCGCGC ATACCGGTAAAGCGGATGGTCTTCCACGAGATCACCGAACCGGCGATCCGCGCCGCCGCCGAGCA CCCCCGCGACCTAGACATCGACCTGGTCGACGCGCAGGAGACCCGGCGCATCCTGGACCGGCTGT ACGGCTACGAAGTCAGCCCAGTGCTGTGGAAGAAGGTCGCCCCCAAGTTGTCGGCGGGCCGGGTG CAGTCGGTGGCCACCCGCATCATCGTGGCGCGCGAACGCGACCGCATGGCGTTCCGCAGCGCGGC CTACTGGGACATCCTTGCCAAGCTGGATGCCAGCGTGTCCGACCCGGACGCCGCGCCGCCCACCT TCAGCGCCCGGCTGACGGCCGTGGCTGGCCGGCGGGTGGCCACTGGCCGCGATTTCGACTCGCTG GGCACGCTGCGCAAAGGCGACGAAGTCATTGTGCTCGACGAGGGGAGCGCGACCGCGTTGGCCGC GGGCCTGGATGGCACGCAGCTGACCGTGGCCTCGGCCGAGGAGAAGCCCTACGCCCGGCGCCCGT ACCCGCCGTTCATGACCTCCACGCTGCAGCAAGAGGCCAGCCGCAAGCTGCGGTTCTCCGCCGAG CGGACGATGAGCATCGCCCAGCGGCTGTACGAAAACGGCTACATCACCTATATGCGTACCGACTC CACCACGCTGTCGGAGTCGGCGATCAACGCCGCACGTACCCAGGCGCGCCAGCTCTACGGCGACG AGTACGTCGCGCCGGCGCCGCGCCAATACACCCGCAAGGTGAAGAACGCCCAGGAAGCGCACGAG GCTATCCGGCCCGCCGGTGAAACGTTTGCCACCCCGGACGCGGTGCGTCGCGAACTCGACGGTCC CAACATTGATGATTTCCGGCTCTATGAGCTGATTTGGCAACGCACCGTAGCCTCGCAGATGGCCG ATGCGCGGGGCATGACGCTGAGCCTGCGGATCACTGGCATGTCGGGGCACCAGGAGGTGGTGTTC TCCGCGACCGGACGCACCTTGACGTTCCCGGGCTTCCTCAAGGCCTACGTGGAGACCGTGGACGA GCTGGTCGGCGGCGAGGCTGACGATGCCGAGCGGCGACTGCCCCATCTGACCCCGGGTCAACGGT TGGACATCGTCGAGTTGACCCCAGACGGCCATGCCACCAACCCGCCGGCCCGCTACACCGAGGCG TCGCTGGTCAAAGCGCTCGAGGAGCTGGGCATCGGCCGCCCGTCGACCTACTCGTCGATCATCAA GACCATCCAGGATCGCGGCTACGTGCACAAGAAGGGCAGTGCACTGGTGCCGTCATGGGTGGCGT TCGCGGTAACCGGTCTGCTCGAGCAGCATTTCGGTCGGCTCGTCGACTACGACTTCACCGCGGCG ATGGAAGACGAGCTCGACGAGATCGCCGCCGGCAACGAGCGCCGCACCAACTGGCTCAACAACTT CTACTTTGGTGGCGATCACGGTGTGCCCGATTCGGTAGCCCGATCGGGTGGCCTCAAGAAGCTTG TCGGGATCAATCTCGAGGGCATCGACGCACGAGAAGTAAACTCTATCAAGCTTTTTGACGACACC CACGGACGCCCCATATATGTTCGGGTGGGCAAGAACGGTCCCTACCTGGAACGTTTGGTGGCCGG CGACACCGGTGAGCCCACGCCGCAGCGGGCCAACCTCAGCGACTCGATTACCCCGGACGAGCTGA CTCTACAGGTGGCCGAAGAGCTCTTTGCCACACCGCAACAGGGACGGACTTTGGGCTTGGACCCA GAAACCGGCCACGAGATCGTGGCCAGGGAAGGCCGGTTTGGGCCGTATGTGACCGAGATCCTGCC GGAGCCTGCGGCTGATGCGGCCGCGGCCGCTCAGGGAGTCAAGAAACGCCAGAAGGCCGCCGGGC CCAAACCGCGCACCGGTTCGTTGCTGCGGAGCATGGACCTACAGACGGTCACCCTCGAAGACGCG CTGAGGCTGCTGTCACTGCCGCGCGTGGTCGGAGTGGACCCCGCCTCGGGTGAGGAGATCACCGC GCAGAACGGGCGCTACGGACCGTATCTAAAGCGCGGCAACGATTCTCGATCACTGGTCACCGAAG ACCAGATATTCACCATCACGCTCGACGAAGCCCTGAAGATCTACGCAGAGCCGAAACGTCGTGGC CGGCAAAGCGCTTCGGCTCCGCCGCTGCGCGAGCTGGGAACAGATCCGGCGTCGGGCAAGCCAAT GGTCATCAAGGACGGCCGATTCGGGCCGTACGTCACCGACGGTGAGACCAATGCCAGCCTGCGTA AGGGCGACGACGTGGCTTCCATAACCGACGAGCGCGCCGCCGAGCTGTTGGCCGATCGCCGAGCC CGGGGTCCGGCAAAACGGCCAGCCAGGAAAGCTGCCCGGAAGGTGCCGGCGAAGAAGGCAGCCAA GCGCGACTAG MycobacteriumtuberculosistopoisomeraseIproteinsequence: SEQIDNO:19 MADPKTKGRGSGGNGSGRRLVIVESPTKARKLASYLGSGYIVESSRGHIRDLPRAASDVPAKYKS QPWARLGVNVDADFEPLYIISPEKRSTVSELRGLLKDVDELYLATDGDREGEAIAWHLLETLKPR IPVKRMVFHEITEPAIRAAAEHPRDLDIDLVDAQETRRILDRLYGYEVSPVLWKKVAPKLSAGRV QSVATRIIVARERDRMAFRSAAYWDILAKLDASVSDPDAAPPTFSARLTAVAGRRVATGRDFDSL GTLRKGDEVIVLDEGSATALAAGLDGTQLTVASAEEKPYARRPYPPFMTSTLQQEASRKLRFSAE RTMSIAQRLYENGYITYMRTDSTTLSESAINAARTQARQLYGDEYVAPAPRQYTRKVKNAQEAHE AIRPAGETFATPDAVRRELDGPNIDDFRLYELIWQRTVASQMADARGMTLSLRITGMSGHQEVVF SATGRTLTFPGFLKAYVETVDELVGGEADDAERRLPHLTPGQRLDIVELTPDGHATNPPARYTEA SLVKALEELGIGRPSTYSSIIKTIQDRGYVHKKGSALVPSWVAFAVTGLLEQHFGRLVDYDFTAA MEDELDEIAAGNERRTNWLNNFYFGGDHGVPDSVARSGGLKKLVGINLEGIDAREVNSIKLFDDT HGRPIYVRVGKNGPYLERLVAGDTGEPTPQRANLSDSITPDELTLQVAEELFATPQQGRTLGLDP ETGHEIVAREGRFGPYVTEILPEPAADAAAAAQGVKKRQKAAGPKPRTGSLLRSMDLQTVTLEDA LRLLSLPRVVGVDPASGEEITAQNGRYGPYLKRGNDSRSLVTEDQIFTITLDEALKIYAEPKRRG RQSASAPPLRELGTDPASGKPMVIKDGRFGPYVTDGETNASLRKGDDVASITDERAAELLADRRA RGPAKRPARKAARKVPAKKAAKRD P.F.TopI Forinformation,cf: http://www.ncbi.nlm.nih.gov/gene/812833 PlasmodiumfalciparumGenesequence(ACCESSIONNC_004326): http://www.ncbi.nlm.nih.qov/nuccore/NC_004326?report=genbank&from= 445981&to=448500&strand=true SEQIDNO:20 1 atgcaatcaatggaaataaatgataataacagtatcaagaatgaaagtacatctgatgat 61 gatatattaattaataaaattaaacaaaacttgggtaataataaatcatgtaattctaga 121 tcttccaaaaaggaatctataaaaaagcaaaagagcaattctgaacttggtataaaaaag 181 aacacaaagaaatcattaggtataaaaaaagaggaagaaaaaaaaaaacaaataagcaaa 241 agaaaaagtaatgaactaaaagaaaaaaataatttgaaagagggaaaaaagaaatatgtg 301 gaaaaaaaatctagaacagtaaaagatgaaaccaagttaacgaatgttataaaaaaagaa 361 actcaaaataataagaaacctaaaaaattacttaaaaaatcagaagaaaattttgaacca 421 ataaatagatggtgggaaaaaatagatgatcaaacagatatacaatggaattatttagaa 491 catcgaggattaatattttcccctccatacgttcaacatcatgtaccaattttttataaa 541 agtataaaaattgaattaaatgcaaaatcagaagaattagctacctattggtgtagtgca 601 attggtagtgattattgtacaaaagaaaagtttatattaaatttttttaaaacatttata 661 aatagtttagaaaatgataatattataaaacaagagaatgaaacgaaattaaaaaaagga 721 gatatatctaattttaagtttattgattttatgccaatcaaagatcatttattaaaatta 781 agagaagaaaagttaaataaaacaaaagaagaaaaagaagaggaaaaaaaaatgagaatg 841 gaaaaagaattaccatatacatatgcgttagttgattggattcgtgaaaagatatcaagt 901 aataaagcagaaccacctgggttatttagaggaagaggagaacatccaaaacaaggttta 961 ttaaaaaaaagaatttttccagaagatgttgtaattaatattagtaaagatgcacctgta 1021 ccacgattatatgataatatgtgtggacataattggggtgatatatatcatgataataaa 1081 gtaacatggttagcttattataaagatagtataaatgatcaaataaaatatactttttta 1141 tctgctcaatcaaaatttaaaggatataaagatcttatgaaatatgaaaatgctcgaaaa 1201 ttaaaatcatgtgttcataaaattagggaagattataaaaataaaatgaaaaataaaaat 1261 attattgataaacaattaggaacagctgtttatttaatagattttctagcattaagagta 1321 ggaggagaaaaagatatcgatgaagaagcagatactgtaggttgttgtagtttaagagta 1381 gaacatattagttttgcacacgatataccttttaaaagtgtagattcaaaagaacaaaaa 1441 acaaatgatgaaaaagtaaataaaataccattaccaacaaatttagaaagtatttcatca 1501 gaagattgttatataactttagattttttaggaaaagatagtatacgatattttaataca 1561 gtcaaaatagataaacaagcatatattaatataataatattttgtaaaaataaaaataga 1621 gatgaaggagtttttgatcaaataacttgttcaaaattaaatgaatatctaaaagaaatt 1681 atgcctactttatcagctaaagtgtttcgtacatataatgcttcaattacattagatcaa 1741 caattaaaaagaataaaagaagtttatggaaaaacaacatattcattatattctggtgaa 1801 acagaattacacaaatcgaaaaaaagaaaatctagccatttaacttcagatacaaatata 1861 ttaagtgatgcaagtgattctactattaatgatgtaaataacgagtatgatgaaaatgga 1921 ataaataaaaaactatcatatgctactactgtaggaaaagaaaatgatgtcgatgataaa 1981 aactcaccaatagaagttgacgtttcaaatataaatgaacttattaatttttacaataat 2041 gcaaatagagaagtagccatattatgtaaccatcaaagaagtattccaaaacaacatgat 2101 acaactatgtcaaaaataaaaaaacaaattgaattatataatgaagatataaaagaatat 2161 aaaaaatatttgcaacatttaaaaaaaaatagtgataaaaaatttatctttgtttcgaaa 2221 gtttctactttagatggaactttaagaccaaataaagtcaaagaaaatatgaaagaagaa 2281 tcttgtaaaaaaaaactaattactcttataaaaaaagttgaattattaaataaccaaatg 2341 aaagtaagagatgataataaaactattgctttaggtacatctaaaattaattatatggat 2401 ccaagaataactgttgctttttgtaaaaaatttgaaatacccatagaaaaagtatttaat 2461 agaagtttaagacttaaatttccttgggccatgtttgctacaaaaaattttacattttaa PlasmodiumfalciparumProteinsequence(ACCESSIONXP_001351663): http://www.ncbi.nlm.nih.gov/protein/XP_001351663.1 SEQIDNO:21 1 mqsmeindnnsiknestsdddilinkikqnlgnnkscnsrsskkesikkqksnselgikk 61 ntkkslgikkeeekkkqiskrksnelkeknnlkegkkkyvekksrtvkdetkltnvikke 121 tqnnkkpkkllkkseenfepinrwwekiddqtdiqwnylehrglifsppyvqhhvpifyk 181 sikielnakseelatywcsaigsdyctkekfilnffktfinslendniikqenetklkkg 241 disnfkfidfmpikdhllklreeklnktkeekeeekkmrmekelpytyalvdwirekiss 301 nkaeppglfrgrgehpkqgllkkrifpedvviniskdapvprlydnmcghnwgdiyhdnk 361 vtwlayykdsindqikytflsaqskfkgykdlmkyenarklkscvhkiredyknkmknkn 421 iidkqlgtavylidflalrvggekdideeadtvgccslryehisfahdipfksvdskeqk 481 tndekvnkiplptnlesissedcyitldflgkdsiryfntvkidkqayiniiifcknknr 541 degvfdqitcsklneylkeimptlsakvfrtynasitldqqlkrikevygkttyslysge 601 telhkskkrksshltsdtnilsdasdstindvnneydenginkklsyattvgkendvddk 661 nspievdvsninelinfynnanrevailcnhqrsipkqhdttmskikkqielynedikey 721 kkylqhlkknsdkkfifvskvstldgtlrpnkvkenmkeesckkklitlikkvellnnqm 781 kvrddnktialgtskinymdpritvafckkfeipiekvfnrslrlkfpwamfatknftf StreptococcusagalactiaeDNAtopoisomeraseIaminoacidsequence (ACCESSIONWP_000246605): SEQIDNO:27 1 matttktstkktskkksatakknlvivespakaktiekylgrnykvvasvghirdlkkss 61 msidfennyepqyinirgkgplindlkkeakkakkvylasdpdregeaiswhlahildld 121 kedrnrvvfneitkdavknafveprqinmdlvdaqqarrvldrivgysispilwkkvkkg 181 lsagrvqsvalkliidreneikafqpeeywtidgsfkkgtrkfnatfygldgkkfklsnn 241 edvktvlkriktdeflvekvekkerprnaplpyttsslqqdaankinfrtrktmmiaqql 301 yeglslgtaghqglitymrtdstrisplaqneaaefitnrfganyskhgnkvknasgaqd 361 aheairpssvnhtpesiakyldkdqlklytliwnrfiasqmtaavfdtmkvnltqngvtf 421 iangsqvkfdgymavyndtdknkmlpdmeegesvkkvntnpeqhftqpparfseaslikt 481 leengvgrpstyaptletiqkryyvklaakrfeptelgeivnsliveffpdivdvtftae 541 megkldeveigkeqwqkiidefykpfekelakaetemekiqikdepagfdcelcgspmvi 601 klgrygkfyacsnfpechntkaitkeigvicpicqkgqvierktkrnrifygcdrypece 661 ftswdkpigrtcpksndflvekkvrgggkqvvcsnekcdyqeekik