Selective detection of oncogenic HPV

• US 8,124,334 B2
• Filed: 07/06/2005
• Issued: 02/28/2012
• Est. Priority Date: 07/06/2004
• Status: Active Grant

• Current Assignee: Enzo Biochem Incorporated
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Donegan, James J., Rabbani, Elazar
• Primary Examiner(s): Babic, Christopher M.
• Application Number:
  US11/175,884
• Publication Number:
  US 20090191538A1
• Time in Patent Office: 2,428 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

Compositions and methods for discriminately detecting the presence of a set of related genes from target organisms while avoiding detection of closely similar genes in non-target organisms. The present invention achieves this objective by a variety of novel nucleic acid constructs and methods. The nucleic acid constructs of the present invention are able to carry out this objective by virtue of the selected sequences of the compositions and by methods of use of such compositions.

Claims

1. A process for detecting the presence of HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in a sample, said process comprising the steps of:

A) providing;

(i) said sample suspected of containing any of said HPV types;

(ii) a set of nucleic acid constructs comprising;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (n), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (n−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (p), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (p−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (s), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (s−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (t), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (t−

1);

B) contacting said set of nucleic acid constructs (ii) with said sample (i) under conditions suitable for hybridization of said constructs (ii) to complementary sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 nucleic acids, if present in said sample; and

C) detecting said hybridization.

2. The process of claim 1 wherein said set of constructs (ii) comprise ribonucleotides and wherein said detecting step C) is carried out by detecting the presence of RNA/DNA hybrids.

3. The process of claim 1, wherein said nucleic acid constructs are labeled.

4. The process of claim 1, wherein nucleic acids in said sample (i) have been subjected to a nucleic acid amplification procedure.

5. The process of claim 1, wherein nucleic acids in said sample have not been amplified.

6. The process of claim 1 wherein said contacting step B) is carried out in situ.

7. The process of claim 1, wherein nucleic acids in said sample have been isolated or purified.

8. The process of claim 1, wherein said contacting step B) and said detecting step C) take place during an amplification procedure.

9. The process of claim 3, wherein said label comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component or a chelating component, a radioisotope, a phosphorescent component, an intercalating component, an energy transfer component, or any combination of the above.

10. The process of claim 4 wherein said amplification procedure has been carried out using at least two set of primers wherein at least one primer of a first set binds to one strand of an HPV nucleic acid and at least one primer of a second set binds to the complementary strand of said HPV nucleic acid.

11. The process of claim 8 wherein said amplification procedure is carried out using at least two set of primers wherein at least one primer of a first set of primers binds to one strand of an HPV nucleic acid and at least one primer of a second set of primers binds to the complementary strand of said HPV nucleic acid.

12. The process of claim 10 or 11 wherein said first set of primers comprises one or more consensus primers and said second set of primers comprises one or more consensus primers, wherein said consensus primers are capable of binding to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68.

13. The process of claim 10 or 11, wherein said first set of primers comprise one or more consensus primers wherein said consensus primers are capable of binding to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 and wherein said second set of primers comprise:

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T−

1).

14. The process of claim 10 or 11, wherein said first set of primers comprise:

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N_F−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P_F−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q_F−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S_F), and the highest number of matches with homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S_F−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T_F−

1)

and said second set of primers comprise;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N_R1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P_R−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q_R−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S_R−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T_R−

1).

15. The process of claim 1, wherein a set of suppressor nucleic acid constructs are also provided wherein said suppressor nucleic acid constructs comprise

a) at least one (1) first suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (g), and the highest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to or less than an integer (g−

1);

b) at least one (1) second suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (h), and the highest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to or less than an integer (h−

1);

c) at least one (1) third suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (j), and the highest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to or less than an integer (j−

1);

d) at least one (1) fourth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (k), and the highest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to or less than an integer (k−

1); and

e) at least one (1) fifth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with the homologous sequence in HPV 6 or HPV 11 is equal to an integer (m) and the highest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to or less than an integer (m−

1); and

wherein said suppressor nucleic acid constructs comprise sequences that are homologous to all or a portion of one or more of the sequences of the nucleic acid constructs (ii).

16. The process of claim 10 or 11, wherein a set of suppressor nucleic acid constructs are also present during said amplification, wherein said suppressor nucleic acid constructs comprise:

a) at least one (1) first suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (g), and the highest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to or less than an integer (g−

1);

b) at least one (1) second suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (h), and the highest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to or less than an integer (h−

1);

c) at least one (1) third suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (j), and the highest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to or less than an integer (j−

1);

d) at least one (1) fourth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (k), and the highest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to or less than an integer (k−

1); and

e) at least one (1) fifth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with the homologous sequence in HPV 6 or HPV 11 is equal to an integer (m) and the highest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to or less than an integer (m−

1); and

wherein said suppressor nucleic acid constructs comprise sequences that are homologous to all or a portion of one or more said primers.

17. The process of claim 4 or 7 wherein said amplification procedure comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

18. A process for detecting the presence of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in a sample, said process comprising the steps of:

A) providing;

i) said sample suspected of containing any of said HPV types;

ii) a first set of nucleic acid constructs comprising;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (n), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (n−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (p), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (p−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (s), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (s−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (t), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (t−

1);

iii) a second set of nucleic acid constructs comprising;

a) at least one (1) first suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (g), and the highest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to or less than an integer (g−

1);

b) at least one (1) second suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (h), and the highest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to or less than an integer (h−

1);

c) at least one (1) third suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (j), and the highest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to or less than an integer (j−

1);

d) at least one (1) fourth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 6 or HPV 11 is equal to an integer (k), and the highest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to or less than an integer (k−

1); and

e) at least one (1) fifth suppressor nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with the homologous sequence in HPV 6 or HPV 11 is equal to an integer (m) and the highest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to or less than an integer (m−

1); and

wherein said suppressor nucleic acid constructs comprise sequences that are homologous to all or a portion of one or more of the sequences of the nucleic acid constructs (ii);

B) contacting said sets of nucleic acid constructs (ii) and (iii) with said sample (i) under conditions suitable for hybridization of said constructs (ii) to complementary sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68, if present in said sample; and

C) detecting said hybridization.

19. The process of claim 18 wherein said first set of constructs (ii) comprise ribonucleotides and said second set of constructs (iii) lack ribonucleotides and wherein said detecting step C) is carried out by detecting the presence of RNA/DNA hybrids.

20. The process of claim 18, wherein said first set of nucleic acid constructs (ii) are labeled.

21. The process of claim 18, wherein said nucleic acids in said sample (i) have been subjected to a nucleic acid amplification procedure.

22. The process of claim 18, wherein nucleic acids in said sample (i) have not been amplified.

23. The process of claim 18, wherein said contacting step B) is carried out in situ.

24. The process of claim 18, wherein nucleic acids in said sample (i) have been isolated or purified.

25. The process of claim 18, wherein said contacting step B) and said detecting step C) are carried out during an amplification procedure.

26. The process of claim 20, wherein said label is selected from biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

27. The process of claim 21 wherein said amplification procedure has been carried out using at least two set of primers wherein at least one primer of a first set of primers binds to one strand of an HPV nucleic acid and at least one strand of a second set of primers binds to the complementary strand of said HPV nucleic acid.

28. The process of claim 25 wherein said amplification procedure is carried out using at least two set of primers wherein at least one primer of a first set of primers binds to one strand of an HPV nucleic acid and at least one primer of a second set of primers binds to the complementary strand of said HPV nucleic acid.

29. The process of claim 27 or 28 wherein said first set of primers comprises one or more consensus primers and said second set of primers comprises one or more consensus primers, wherein said consensus primers are capable of binding to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68.

30. The process of claim 27 or 28, wherein said first set of primers comprise one or more consensus primers wherein said consensus primers are capable of binding to HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 and wherein said second set of primers comprise:

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T−

1).

31. The process of claim 27 or 28, wherein said first set of primers comprise:

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N_F−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P_F−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q_F−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S_F−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T_F), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T_F−

1) and said second set of primers comprise;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N_R1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P_R−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q_R−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S_R−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T_R), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T_R−

1).

32. The process of claim 21 or 25 wherein said amplification procedure comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

33. A method of detecting the presence of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in a sample comprising the steps of:

A) providing;

i) a sample;

ii) a first set of nucleic acid constructs comprising sequences complementary to sequences of a strand of HPV nucleic acid wherein said set comprises;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (n), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (n−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (p), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (p−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (s), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer, (s−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (t), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (t−

1);

iii) a second set of nucleic acid constructs comprising sequences substantially identical to sequences of said strand of HPV nucleic acid; and

iv) reagents appropriate for carrying out extension of said nucleic acid constructs;

B) contacting said first set of nucleic acid constructs with said sample under conditions suitable for hybridization of said nucleic acid constructs to complementary sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 if present in the sample;

C) extending one or more of said first set of nucleic acid constructs using the sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 as templates;

D) contacting said second set of nucleic acid constructs with said extended first nucleic acid constructs from step (D);

E) extending one or more of said second set of nucleic acid constructs using the extended nucleic acid construct from step (E) as templates;

F) repeating one or more of the preceding steps; and

G) detecting extension products synthesized by using HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 as templates and thereby determining the presence of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in the sample.

34. The method of claim 33, wherein said second set comprises a set of nucleic acid constructs wherein:

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T−

1).

35. The method of claim 33 wherein said second set comprises HPV consensus primers.

36. The method of claim 33, 34 or 35 wherein said extension step (C) is carried out by a DNA polymerase.

37. The method of claim 36 wherein said DNA polymerase is selected from one or more of the group comprising Taq DNA polymerase, Klenow Fragment, DNA polymerase I, Bst DNA polymerase, Bca DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, Tfl DNA polymerase, Pfx DNA polymerase, Elongase, Herculase, Taq Plus, Deep-vent Polymerase, Vent DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Bca DNA polymerase, Thermal Ace™

polymerase, or any mutants or modified versions of the foregoing.

38. The method of claim 36 wherein said DNA polymerase is a Reverse Transcriptase.

39. The method of claim 38 wherein said Reverse Transcriptase comprises HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, AMV reverse transcriptase, MMLV reverse transcriptase, RSV reverse transcriptase, ALV reverse transcriptase, MuLV reverse transcriptase, Sensiscript, SuperScript™

, Thermo Script™

or Omniscript.

40. The method of claim 39 wherein said Reverse Transcriptase has been modified such that it is RnaseH⁻

.

41. The method of claim 33 or 34, wherein said extension step (C) is carried out by ligase.

42. The method of claim 41, wherein said ligase comprises T4 DNA ligase, T4 RNA ligase, Pyrococcus furiosus DNA ligase, Escherichia coli DNA ligase, Taq DNA ligase, recombinant Human ligase I, recombinant Human ligase II, recombinant Human ligase III or recombinant Human ligase IV.

43. The method of claim 33, 34 or 35 wherein said second set is used for extension prior to said first set.

44. The method of claim 33, 34 or 35 wherein one or more of said nucleic acid constructs comprise sequences for RNA promoters.

45. The method of claim 44 wherein said process comprises a further step of transcription from said RNA promoters.

46. The method of claim 44 wherein said RNA promoters comprise T3, T7 or SP6.

47. The method of claim 33, wherein said reagents (iv) comprise one or more modified nucleotides or nucleotide analogues.

48. The method of claim 47, wherein said modified nucleotides are labeled or unlabeled.

49. The method of claim 47, wherein said nucleotide analogues are labeled or unlabeled.

50. The method of claim 48 or 49, wherein said labeled modified nucleotides or labeled nucleotide analogues comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

51. The method of claim 50, wherein said detections step (G) is carried out by the detection of the presence of extension products labeled by incorporation of said labeled modified nucleotides or labeled nucleotide analogues.

52. The method of claim 33 or 34, wherein said detection step (G) is carried out by hybridization with one or more labeled probes.

53. The method of claim 52, wherein said one or more labeled probes comprise labeled nucleotides or labeled nucleotide analogues.

54. The method of claim 53, wherein said labeled nucleotides or labeled nucleotide analogues comprise biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

55. The method of claim 52, wherein said labeled probe comprises sequences from HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 or any combination thereof.

56. The method of claim 52, wherein said labeled probe comprises a set of probes wherein:

a) at least one (1) first labeled probe comprises a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (PRn), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRn−

1);

b) at least one (1) second labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (PRp), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRp−

1);

c) at least one (1) third labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (PRq), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRq−

1);

d) at least one (1) fourth labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (PRs), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRs−

1); and

e) at least one (1) fifth labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (PRt), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRt−

1).

57. The method of claim 56 wherein at least one of said first labeled probe, said second labeled probe, said third nucleic acid probe, said fourth labeled probe and said fifth labeled probe comprises the same sequence.

58. The method of claim 33, 34 or 35, wherein said detection step (G) is carried out by the presence of an intercalating agent that indicates the synthesis of extension products.

59. The method of claim 33, 34 or 35, wherein said method comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

60. A method of detecting the presence of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in a sample comprising the steps of:

A) providing;

i) a sample;

ii) a first set of nucleic acid constructs comprising sequences complementary to sequences of a strand of HPV nucleic acid wherein said set comprises;

a) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (n), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (n−

1);

b) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (p), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (p−

1);

c) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (q−

1);

d) at least one (1) fourth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (s), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (s−

1); and

e) at least one (1) fifth nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (t), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (t−

1);

iii) a second set of nucleic acid constructs comprising sequences substantially identical to sequences of said strand of HPV nucleic acid wherein said second set comprises a set of nucleic acid constructs wherein;

a) at least one (1) first nucleic acid construct comprises a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (N), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (N−

1);

b) at least one (1) second nucleic acid construct comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (P), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (P−

1);

c) at least one (1) third nucleic acid construct comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (Q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (Q−

1);

d) at least one (1) fourth nucleic acid construct comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (S), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (S−

1); and

e) at least one (1) fifth nucleic acid construct comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (T), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (T−

1); and

iv) reagents appropriate for carrying out extension of said nucleic acid constructs;

B) contacting said first set of nucleic acid constructs with said sample under conditions suitable for hybridization of said nucleic acid constructs to complementary sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 if present in the sample;

C) extending one or more of said first set of nucleic acid constructs using the sequences of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 as templates;

D) contacting said second set of nucleic acid constructs with said extended first nucleic acid constructs from step (D);

E) extending one or more of said second set of nucleic acid constructs using the extended nucleic acid construct from step (E) as templates;

F) repeating one or more of the preceding steps; and

G) detecting extension products synthesized by using HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 as templates and thereby determining the presence of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 or 68 in the sample.

61. The method of claim 60, wherein said extension step (C) is carried out by a DNA polymerase.

62. The method of claim 61 wherein said DNA polymerase comprises Taq DNA polymerase, Klenow Fragment, DNA polymerase I, Bst DNA polymerase, Bca DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, Tfl DNA polymerase, Pfx DNA polymerase, Elongase, Herculase, Taq Plus, Deep-vent Polymerase, Vent DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Bca DNA polymerase, Thermal Ace™

polymerase or any mutants or modified versions of the foregoing.

63. The method of claim 61, wherein said DNA polymerase is a Reverse Transcriptase.

64. The method of claim 63, wherein said Reverse Transcriptase comprises HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, AMV reverse transcriptase, MMLV reverse transcriptase, RSV reverse transcriptase, ALV reverse transcriptase, MuLV reverse transcriptase, Sensiscript, SuperScript™

, Thermo Script™

or Omniscript.

65. The method of claim 63, wherein said Reverse Transcriptase has been modified such that it is RnaseH⁻

.

66. The method of claim 60, wherein said extension step (C) is carried out by ligase.

67. The method of claim 66, wherein said ligase comprises T4 DNA ligase, T4 RNA ligase, Pyrococcus furiosus DNA ligase, Escherichia coli DNA ligase, Taq DNA ligase, recombinant Human ligase I, recombinant Human ligase II, recombinant Human ligase III or recombinant Human ligase IV.

68. The method of claim 60, wherein one or more of said nucleic acid constructs comprises sequences for RNA promoters.

69. The method of claim 68 wherein said method comprises a further step of transcription from said RNA promoters.

70. The method of claim 68 wherein said RNA promoters comprise T3, T7 or SP6.

71. The method of claim 60, wherein said reagents (iv) comprise one or more modified nucleotides or nucleotide analogues.

72. The method of claim 71, wherein said modified nucleotides are labeled or unlabeled.

73. The method of claim 62, wherein said nucleotide analogues are labeled or unlabeled.

74. The method of claim 63 or 64, wherein said labeled modified nucleotides or labeled nucleotide analogues comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

75. The method of claim 60, wherein said detections step (G) is carried out by the detection of the presence of extension products labeled by incorporation of said labeled modified nucleotides or labeled nucleotide analogues.

76. The method of claim 60, wherein said detection step (G) is carried out by hybridization with one or more labeled probes.

77. The method of claim 76, wherein said one or more labeled probes comprise labeled nucleotides or labeled nucleotide analogues.

78. The method of claim 77, wherein said labeled nucleotides or labeled nucleotide analogues comprise biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination of the foregoing.

79. The method of claim 76, wherein said labeled probe comprises sequences from HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68 or any combination thereof.

80. The method of claim 76, wherein said labeled probe comprises a set of probes wherein:

a) at least one (1) first labeled probe comprises a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16, 33, 52 or 58 is equal to an integer (PRn), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRn−

1);

b) at least one (1) second labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 31 or 35 is equal to an integer (PRp), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRp−

1);

c) at least one (1) third labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18, 45 or 59 is equal to an integer (PRq), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRq−

1);

d) at least one (1) fourth labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 39 or 68 is equal to an integer (PRs), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRs−

1); and

e) at least one (1) fifth labeled probe comprises a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or 56 is equal to an integer (PRt), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (PRt−

1).

81. The method of claim 80 wherein at least one of said first labeled probe, said second labeled probe, said third nucleic acid probe, said fourth labeled probe and said fifth labeled probe comprises the same sequence.

82. The method of claim 60 wherein at least one of said first nucleic acid construct, said second nucleic acid construct, said third nucleic acid probe, said fourth labeled probe and said fifth labeled probe of said first set comprises the same sequence.

83. The method of claim 60 wherein at least one of said first nucleic acid construct, said second nucleic acid construct, said third nucleic acid probe, said fourth labeled probe and said fifth labeled probe of said second set comprises the same sequence.

84. The method of claim 60 wherein one or more of said nucleic acid constructs comprises a label.

85. The method of claim 84 wherein said label comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination of the foregoing.

86. The method of claim 60, wherein said method comprises a nucleic acid amplification process.

87. The method of claim 84, wherein said amplification process comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

88. A method of detecting the presence of HPV which comprises:

a) providing;

(i) a sample that may contain nucleic acids from one or more HPV types;

(ii) two sets of primers wherein one or more nucleic acid constructs of a first set of primers are complementary to sequences in one strand of HPV 16, 18, 45, 51, 52 and 56 and wherein one or more nucleic acid constructs of a second set of primers are complementary to the other strand of HPV 16, 18, 45, 51, 52 and 56 and wherein the primers of said first and second sets are complementary to more than one of said HPV types; and

(iii) reagents for carrying out a nucleic acid amplification reaction;

b) carrying out an amplification reaction under conditions where;

A) sequences from HPV 16, 18, 45, 51, 52 or 56 are amplified; and

;

B) if present in said sample, sequences from HPV 6 and HPV 11 are not amplified; and

c) determining the presence of amplified product and thereby the presence of HPV.

89. The method of claim 88, wherein said amplification reaction comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

90. The method of claim 88 wherein said primers (i), said reagents (ii) or both said primers (i) and said reagents (ii) comprise a label.

91. The method of claim 90 wherein said label is attached to a nucleotide.

92. The method of claim 90 or 91, wherein said label comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination of the foregoing.

93. The method of claim 90 or 91 wherein said label comprises an intercalating dye.

94. A method of detecting the presence of HPV which comprises:

a) providing;

(i) a sample that may contain nucleic acids from one or more HPV types;

(ii) reagents for carrying out a nucleic acid reaction; and

(iii) two sets of primers wherein a first set of primers is complementary to one strand of an HPV nucleic acid and a second set of primers is complementary to the other strand of said HPV nucleic acid and wherein said first set comprises;

A) at least one (1) first nucleic acid construct comprising a nucleic acid sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 16 or HPV 52 is equal to an integer (n), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (n−

1);

B) at least one (1) second nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 18 or HPV 45 or is equal to an integer (q), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (q−

1); and

C) at least one (1) third nucleic acid construct comprising a sequence, wherein the lowest number of matches of said sequence with a homologous sequence in HPV 51 or HPV 56 is equal to an integer (t), and the highest number of matches with a homologous sequence in HPV 6 or HPV 11 is equal to or less than an integer (t−

1); and

said second set comprises one or more nucleic acid constructs complementary to sequences in HPV 16, 18, 45, 51, 52 or 56;

b) carrying out an amplification reaction under conditions where A) sequences from HPV 16, 18, 45, 51, 52 or 56 are amplified and B) sequences from HPV 6 and HPV 11 are not amplified; and

c) determining the presence of amplified product and thereby the presence of HPV.

95. The method of claim 94, wherein said amplification reaction comprises the polymerase chain reaction, the ligase chain reaction, the GAP-LCR method, the 3SR reaction, the NASBA reaction, the Transcription Mediated Amplification reaction, the Strand Displacement Amplification reaction, or the isothermal hairpin amplification reaction.

96. The method of claim 94, wherein said primers (i), said reagents (ii) or both said primers (i) and said reagents (ii) comprise a label.

97. The method of claim 96 wherein said label is attached to a nucleotide.

98. The method of claim 96 or 97, wherein said label comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination of the foregoing.

99. The method of claim 96 or 97 wherein said label comprises an intercalating dye.

100. A process for preparing one or more copies of a target nucleic acid after alteration of primer binding sites, comprising the steps of:

(A) providing;

(i) a target nucleic acid to be copied; and

(ii) at least one first forward primer and at least one second forward primer wherein;

(a) said first forward primer comprises one or more selective degenerate bases;

(b) said first forward primer comprises sequences substantially complementary to a first region of said target nucleic acid;

(c) said second forward primer comprises sequences substantially complementary to a nucleic acid formed by using said first forward primer as a template; and

(d) said second forward primer lacks said one or more degenerate bases in said first forward primer;

(iii) one or more reverse primers wherein said reverse primers comprise sequences substantially identical to a second region of said target nucleic acid; and

(iv) means for template-dependent nucleic acid synthesis;

(B) contacting said target nucleic acid with said first forward primer;

(C) extending said first forward primer by means of a template-dependent polymerase to form a first copy; and

(D) rendering a reverse primer binding sequence available in said first copy;

(E) contacting said first copy with said reverse primer;

(F) extending said reverse primer using said first copy as a template wherein at least one of the nucleotides incorporated into a site opposite a degenerate base in said first forward primer is different from the nucleotide in the target nucleic acid used as a template in step (B) thereby forming an altered forward primer binding sequence in said second copy;

(G) rendering said altered forward primer binding sequence available in said second copy;

(H) contacting said second copy with said second forward primer;

(I) extending said second forward primer using said second copy as a template, thereby producing a third copy; and

(J) optionally continuing a further series of rendering, contacting and extension steps and thereby providing one or more copies of said target nucleic acid with an altered primer binding site.

101. The process of claim 100, wherein said one or more reverse primers comprises at least one first reverse primer and at least one second reverse primer wherein:

(a) said first reverse primer comprises one or more selective degenerate bases;

(b) said first reverse primer comprises sequences substantially identical to sequences of a second region of said nucleic acid;

(c) said second reverse primer comprises sequences substantially complementary to a nucleic acid formed by using said first reverse primer as a template; and

(d) said second reverse primer lacks said one or more degenerate bases in said first reverse primer.

102. The process of claim 100 or 101, wherein a further series of rendering, contacting and extending steps are carried to provide additional copies of said target nucleic acids with altered primer binding sequences.

103. The process of claim 102, wherein after a series of rendering, contacting and extending steps, the conditions of contacting and extension are changed such that the target nucleic acid provided in said providing step (i) is less efficiently used as a target for extension events by said first forward primer or said first reverse primer, compared to the use of said extension products by said second forward primer or said second reverse primer.

104. The process of claim 100 wherein said second forward primer comprises one or more additional bases at its 3′

end compared to said first forward primer.

105. The process of claim 101 wherein said second reverse primer comprises one or more additional bases at its 3′

end compared to said first reverse primer.

106. The process of claim 100 or 101 wherein said selective degenerate base in said first forward primer or said first reverse primer comprises Inosine, “

K”

or a combination of the preceding.

107. The process of claim 106 wherein said second forward primer or said second reverse primer comprises a G in the position occupied by Inosine in said first forward primer or said second forward primer.

108. The process of claim 106 wherein said second forward primer or said second reverse primer comprises an A in the position occupied by “

K”

in said first forward primer or said second forward primer.

109. The process of claim 100 or 101, wherein said first forward primer, said second forward primer, said first reverse primer, said second reverse primer or any combination thereof additionally comprise one or more degenerate bases wherein said additional degenerate base lacks a substantial preference for a particular base as a substrate for incorporation when said additional base is used as a template.

110. The process of claim 109 wherein said degenerate base lacking a substantial preference for a particular base as a substrate for incorporation is selected from the group comprising 8-oxo-guanosine and “

P”

.

111. The process of claim 100, 101 or 102, further comprising the step of detecting the presence of said altered priming binding sequence wherein said detection is carded out by measurement of the binding of a labeled probe wherein said labeled probe is complementary to said altered prime binding sequence.

112. The process of claim 111, wherein said labeled probes comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

113. The process of claim 100, 101 or 102, wherein one or more labeled nucleotides or labeled nucleotide analogues are incorporated.

114. The process of claim 113, wherein said labeled nucleotides or labeled nucleotide analogues comprises one or more labels selected from the group consisting of biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

115. The process of claim 100, 101 or 102, wherein one or more of said rendering steps comprises thermal denaturation, secondary structure formation, enzyme digestion, strand displacement or any combination thereof.

116. The process of claim 115 wherein said enzyme comprises RnaseH or a restriction enzyme.

117. The process of claim 102, further comprising the step of detecting the presence of target nucleic acid sequences other than said forward primer binding sequence, said altered forward primer binding sequence, said reverse primer binding sequence and said altered reverse primer binding sequence.

118. The process of claim 100, wherein said means (iv) for template-dependent nucleic acid synthesis comprises one or more members selected from the group consisting Taq DNA polymerase, Klenow Fragment, DNA polymerase I, Bst DNA polymerase, Bca DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, Tfl DNA polymerase, Pfx DNA polymerase, Elongase, Herculase, Taq Plus, Deep-vent Polymerase, Vent DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Bca DNA polymerase, Thermal Ace™

polymerase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, AMV reverse transcriptase, MMLV reverse transcriptase, RSV reverse transcriptase, ALV reverse transcriptase, MuLV reverse transcriptase, Sensiscript, SuperScript™

, Thermo Script™

, Omniscript or any mutants or modified versions of the foregoing.

119. The process of claim 100, 101 or 102, wherein said target nucleic acid is RNA or DNA.

120. The process of claim 100, 101 or 102, wherein said target nucleic acid comprises sequences of Human Papilloma Virus.

121. The process of claim 100, 101 or 102 wherein said rendering, contacting and extension steps take place during a polymerase chain reaction, a 3SR reaction, a NASBA reaction, a Transcription Mediated Amplification reaction, a Strand Displacement Amplification reaction or an isothermal hairpin amplification reaction.

122. A process for preparing one or more complementary copies of a target nucleic acid with altered primer binding site comprising the steps of:

(A) providing;

(i) a target nucleic acid to be copied; and

(ii) at least one first forward primer and at least one second forward primer wherein;

(a) said first forward primer comprises one or more selective degenerate bases;

(b) said first forward primer comprises sequences substantially complementary to a first region of said target nucleic acid;

(c) said second forward primer comprises sequences substantially complementary to a nucleic acid formed by using said first forward primer as a template;

(d) said second forward primer comprises a non-degenerate base in at least one position occupied by said selective degenerate base in said first forward primer; and

(e) said non-degenerate base is the complement of the preferred base that is incorporated when said selective degenerate base is used in a template;

(iii) one or more reverse primers wherein said reverse primers comprise sequences substantially identical to a second region of said target nucleic acid; and

(iv) means for template-dependent nucleic acid synthesis;

(B) contacting said target nucleic acid with said first forward primer;

(C) extending said first forward primer by means of a template-dependent polymerase to form a first copy;

(D) rendering a reverse primer binding sequence available in said first copy;

(E) contacting said first copy with said reverse primer;

(F) extending said reverse primer using said first copy as a template wherein at least one of the nucleotides incorporated into a site opposite a degenerate base in said first forward primer is different from the nucleotide in the target nucleic acid used as a template in step (b) thereby forming an altered forward primer binding sequence in said second copy;

(G) rendering said altered forward primer binding sequence available in said second copy;

(H) contacting said second copy with said second forward primer;

(I) extending said second forward primer using said second copy as a template, thereby providing a third copy of said target nucleic acid; and

(J) optionally continuing a further series of rendering, contacting and extension steps and thereby providing one or more copies of said target nucleic acid with an altered primer binding site.

123. The process of claim 122, wherein said one or more reverse primers comprises at least one first reverse primer and at least one second reverse primer wherein:

(a) said first reverse primer comprises one or more selective degenerate bases;

(b) said first reverse primer comprises sequences substantially identical to sequences of a second region of said nucleic acid;

(c) said second reverse primer comprises sequences substantially complementary to a nucleic acid formed by using said first reverse primer as a template; and

(d) said second reverse primer lacks said one or more degenerate bases in said first reverse primer.

124. The process of claim 122 or 123, wherein a further series of rendering, contacting and extending steps are carried to provide additional copies of said target nucleic acids with altered primer binding sequences.

125. The process of claim 124, wherein after a series of rendering, contacting and extending steps, the conditions of contacting and extension are changed such that the target nucleic acid provided in said providing step (i) is less efficiently used as a target for extension events by said first forward primer or said first reverse primer, compared to the use of said extension products by said second forward primer or said second reverse primer.

126. The process of claim 122 wherein said second forward primer comprises one or more additional bases at its 3′

end compared to said first forward primer.

127. The process of claim 122 wherein said second reverse primer comprises one or more additional bases at its 3′

end compared to said first reverse primer.

128. The process of claim 122 or 123 wherein said selective degenerate base in said first forward primer or said first reverse primer comprises Inosine, “

K”

or a combination of the preceding.

129. The process of claim 128 wherein said second forward primer or said second reverse primer comprises a G in the position occupied by Inosine in said first forward primer or said second forward primer.

130. The process of claim 128 wherein said second forward primer or said second reverse primer comprises an A in the position occupied by “

K”

in said first forward primer or said second forward primer.

131. The process of claim 122 or 123, wherein said first forward primer, said second forward primer, said first reverse primer, said second reverse primer or any combination thereof additionally comprise one or more degenerate bases wherein said additional degenerate base lacks a substantial preference for a particular base as a substrate for incorporation when said additional base is used as a template.

132. The process of claim 131 wherein said degenerate base lacking a substantial preference for a particular base as a substrate for incorporation is selected from the group comprising 8-oxo-guanosine and “

P”

.

133. The process of claim 122, 123 or 124, further comprising the step of detecting the presence of said altered priming binding sequence wherein said detection is carried out by measurement of the binding of a labeled probe wherein said labeled probe is complementary to said altered prime binding sequence.

134. The process of claim 133, wherein said labeled probes comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

135. The process of claim 122, 123 or 124, wherein one or more labeled nucleotides or labeled nucleotide analogues are incorporated.

136. The process of claim 135, wherein said labeled nucleotides or labeled nucleotide analogues comprises one or more labels selected from the group consisting of biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

137. The process of claim 122, 123 or 124, wherein one or more of said rendering steps comprises thermal denaturation, secondary structure formation, enzyme digestion, strand displacement or any combination thereof.

138. The process of claim 124 wherein said enzyme comprises RnaseH or a restriction enzyme.

139. The process of claim 124, further comprising the step of detecting the presence of target nucleic acid sequences other than said forward primer binding sequence, said altered forward primer binding sequence, said reverse primer binding sequence and said altered reverse primer binding sequence.

140. The process of claim 122, wherein said means (iv) for template-dependent nucleic acid synthesis comprises one or more members selected from the group consisting Taq DNA polymerase, Klenow Fragment, DNA polymerase I, Bst DNA polymerase, Bca DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, Tfl DNA polymerase, Pfx DNA polymerase, Elongase, Herculase, Taq Plus, Deep-vent Polymerase, Vent DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Bca DNA polymerase, Thermal Ace™

polymerase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, AMV reverse transcriptase, MMLV reverse transcriptase, RSV reverse transcriptase, ALV reverse transcriptase, MuLV reverse transcriptase, Sensiscript, SuperScript™

, Thermo Script™

, Omniscript or any mutants or modified versions of the foregoing.

141. The process of claim 122, 123 or 124, wherein said target nucleic acid is RNA or DNA.

142. The process of claim 122, 123 or 124, wherein said target nucleic acid comprises sequences of Human Papilloma Virus.

143. The process of claim 122, 123 or 124, wherein said rendering, contacting and extension steps take place during a polymerase chain reaction, a 3SR reaction, a NASBA reaction, a Transcription Mediated Amplification reaction, a Strand Displacement Amplification reaction or an isothermal hairpin amplification reaction.

144. A process for preparing one or more copies of a target nucleic acid comprising the steps of:

(A) providing;

(i) a target nucleic acid to be copied; and

(ii) at least one first forward primer and at least one second forward primer wherein;

(A) said first forward primer comprises one or more selective degenerate bases;

(B) said first forward primer comprise sequences substantially complementary to sequences of a first region of said nucleic acid;

(C) said second forward primer comprise sequences substantially complementary to a nucleic acid formed by using said first forward primer as a template; and

(D) said second forward primer comprises a non-degenerate base in at least one position occupied by said selective degenerate base in said first forward primer and wherein said non-degenerate base is the complement of the preferred base that is incorporated when said selective degenerate base is used in a template;

(iii) one or more reverse primers wherein said reverse primers comprise sequences substantially identical to a second region of said target nucleic acid; and

(iv) means for template-dependent nucleic acid synthesis;

(B) contacting said target nucleic acid with said first forward primer and binding said first forward primer to a forward primer binding sequence;

(C) extending said first forward primer by means of a template-dependent polymerase to form a first copy;

(D) rendering a reverse primer binding sequence available in said first copy;

(E) contacting said first copy with said reverse primer and binding said reverse primer to said reverse primer binding sequence;

(F) extending said reverse primer using said first copy as a template wherein said preferred nucleotide is inserted opposite said degenerate base;

(G) rendering a forward primer binding sequence available in said second copy;

(H) contacting said second copy with said second forward primer and binding said second forward primer to said forward primer binding sequence in said second copy; and

(I) extending said second forward primer using said second copy as a template thereby providing a copy of said target nucleic acid; and

(J) optionally continuing a further series of rendering, contacting and extension steps and thereby providing one or more copies of said target nucleic acid.

145. The process of claim 144, wherein said one or more reverse primers comprises at least one first reverse primer and at least one second reverse primer wherein:

(a) said first reverse primer comprises one or more selective degenerate bases;

(b) said first reverse primer comprises sequences substantially identical to sequences of a second region of said nucleic acid;

(c) said second reverse primer comprises sequences substantially complementary to a nucleic acid formed by using said first reverse primer as a template; and

(d) said second reverse primer lacks said one or more degenerate bases in said first reverse primer.

146. The process of claim 144 or 145, wherein a further series of rendering, contacting and extending steps are carried to provide additional copies of said target nucleic acids with altered primer binding sequences.

147. The process of claim 146, wherein after a series of rendering, contacting and extending steps, the conditions of contacting and extension are changed such that the target nucleic acid provided in said providing step (i) is less efficiently used as a target for extension events by said first forward primer or said first reverse primer, compared to the use of said extension products by said second forward primer or said second reverse primer.

148. The process of claim 144, wherein said second forward primer comprises one or more additional bases at its 3′

end compared to said first forward primer.

149. The process of claim 145, wherein said second reverse primer comprises one or more additional bases at its 3′

end compared to said first reverse primer.

150. The process of claim 144 or 145, wherein said selective degenerate base in said first forward primer or said first reverse primer comprises Inosine, “

K”

or a combination of the preceding.

151. The process of claim 150 wherein said second forward primer or said second reverse primer comprises a G in the position occupied by Inosine in said first forward primer or said second forward primer.

152. The process of claim 150 wherein said second forward primer or said second reverse primer comprises an A in the position occupied by “

K”

in said first forward primer or said second forward primer.

153. The process of claim 144 or 145, wherein said first forward primer, said second forward primer, said first reverse primer, said second reverse primer or any combination thereof additionally comprise one or more degenerate bases wherein said additional degenerate base lacks a substantial preference for a particular base as a substrate for incorporation when said additional base is used as a template.

154. The process of claim 153 wherein said degenerate base lacking a substantial preference for a particular base as a substrate for incorporation is selected from the group comprising 8-oxo-guanosine and “

P”

.

155. The process of claim 144, 145 or 146, further comprising the step of detecting the presence of said altered priming binding sequence wherein said detection is carried out by measurement of the binding of a labeled probe wherein said labeled probe is complementary to said altered prime binding sequence.

156. The process of claim 155, wherein said labeled probes comprises biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

157. The process of claim 144, 145 or 146, wherein one or more labeled nucleotides or labeled nucleotide analogues are incorporated.

158. The process of claim 157, wherein said labeled. nucleotides or labeled nucleotide analogues comprises one or more labels selected from the group consisting of biotin, iminobiotin, an electron dense component, a magnetic component, a hormone component, a metal-containing component, a fluorescent component, a chromogenic component, a chemiluminescent component, an antigen, a hapten, an antibody component, a chelating component or any combination thereof.

159. The process of claim 144, 145 or 146, wherein one or more of said rendering steps comprises thermal denaturation, secondary structure formation, enzyme digestion, strand displacement or any combination thereof.

160. The process of claim 159 wherein said enzyme comprises RnaseH or a restriction enzyme.

161. The process of claim 146, further comprising the step of detecting the presence of target nucleic acid sequences other than said forward primer binding sequence, said altered forward primer binding sequence, said reverse primer binding sequence and said altered reverse primer binding sequence.

162. The process of claim 144, wherein said means (iv) for template-dependent nucleic acid synthesis comprises Taq DNA polymerase, Klenow Fragment, DNA polymerase I, Bst DNA polymerase, Bca DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, Tfl DNA polymerase, Pfx DNA polymerase, Elongase, Herculase, Taq Plus, Deep-vent Polymerase, Vent DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, Bca DNA polymerase, Thermal Ace™

polymerase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, AMV reverse transcriptase, MMLV reverse transcriptase, RSV reverse transcriptase, ALV reverse transcriptase, MuLV reverse transcriptase, Sensiscript, SuperScript™

, Thermo Script™

, Omniscript, any Mutants or modified versions of the foregoing or any combination of the foregoing.

163. The process of claim 144, 145 or 146, wherein said target nucleic acid is RNA or DNA.

164. The process of claim 144, 145 or 146, wherein said target nucleic acid comprises sequences of Human Papilloma Virus.

165. The process of claim 144, 145 or 146, wherein said rendering, contacting and extension steps take place during a polymerase chain reaction, a 3SR reaction, a NASBA reaction, a Transcription Mediated Amplification reaction, a Strand Displacement Amplification reaction or an isothermal hairpin amplification reaction.