Nucleic acid primer/construct compositions

• US 8,133,989 B2
• Filed: 11/27/2002
• Issued: 03/13/2012
• Est. Priority Date: 06/24/1998
• Status: Expired due to Fees

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Diagnostics, Inc.
• Inventors: Rabbani, Elazar, Stavrianopoulos, Jannis G., Donegan, James J., Coleman, Jack, Walner, Marleen
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US10/307,108
• Publication Number:
  US 20030165939A1
• Time in Patent Office: 3,394 Days
• Field of Search: None
• US Class Current: 536/24.3
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In non-linear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-termination labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.

Claims

1. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence comprising

(i) a first primer comprising at least two segments:

(A) a first segment (i) being substantially complementary to a first portion of said HBV nucleic acid sequence and (ii) capable of template dependent extension such that a first primer extension product is produced when said HBV nucleic acid sequence is used as a template; and

(B) a second segment being (i) substantially non-identical and substantially non-complementary to said first segment, and (ii) substantially identical to a second portion of said hepatitis B virus (HBV) nucleic acid sequence, such that said second segment of said first primer is capable of hybridizing to said first primer extension product and

(ii) a second primer comprising two regions;

(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto, and (2) capable of template-dependent extension to produce a second primer extension product when said first primer extension product is used as a template; and

(B) a second region being (1) substantially non-identical and substantially non-complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of said second primer to said first primer extension product under isothermal or limited cycling conditions.

2. The composition of claim 1, wherein said first primer or said second primer comprises at least one modified nucleotide or nucleotide analog.

3. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence comprising

(i) at least one first initial primer comprising two segments;

(A) a first segment (1) being sufficiently complementary to a first portion of said HBV nucleic acid sequence to hybridize thereto, and (2) capable of template dependent extension to produce a first primer extension product when said HBV nucleic acid sequence is used as a template, and

(B) a second segment being (1) substantially non-identical and substantially non-complementary to said first segment of said first initial primer, and (2) substantially identical to a second portion of said HBV nucleic acid sequence, such that said second segment of said first initial primer is capable of hybridizing to said first primer extension product, thereby providing for subsequent binding of a first segment of a second copy of said first initial primer to said first portion of said hepatitis B virus (HBV) nucleic acid sequence under isothermal or limited cycling conditions, such that a second copy of said first primer extension product is produced and displaces said first primer extension product; and

(ii) at least one second initial primer comprising two regions;

(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto and (2) capable of template-dependent extension to produce a second primer extension product when said first extension product is used as a template, and

(B) a second region being (1) substantially nonidentical and substantially non- complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of said second initial primer to said first portion of said first primer extension product under isothermal or limited cycling conditions.

4. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest comprising

(i) at least one first initial primer, said first initial primer comprising:

(A) a first segment (1) being sufficiently complementary to a first portion of said hepatitis B virus (HBV) nucleic acid sequence to hybridize thereto, and (2) capable of template dependent extension to produce a first primer extension product when said HBV nucleic acid sequence is used as a template, and

(B) a second segment being (1) substantially non-identical and substantially non-complementary to said first segment of said first initial primer, and (2) substantially identical to a second portion of said HBV nucleic acid sequence, such that said second segment of said first initial primer is capable of hybridizing to said first primer extension product, thereby providing for subsequent binding of a first segment of a second copy of said first initial primer to said first portion of said (HBV) nucleic acid sequence under isothermal or limited cycling conditions, such that a second copy of said first primer extension product is produced and displaces said first primer extension product; and

(ii) at least one second initial primer comprising sequences complementary to said first primer extension product, wherein the 5′

end of the second initial primer is joined to the 5′

end of the second segment of the first initial primer.

5. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest under isothermal conditions comprising a primer, wherein said primer comprises at least three segments:

(A)two first segments each comprising an extendable 3′

end, each of which is complementary to a different strand of a double-stranded HBV nucleic acid sequence; and

(B) one second segment that is complementary to one strand of the HBV nucleic acid sequence, wherein the 5′

end of the second segment is joined to the 5′

end of one of the first segments.

6. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest under isothermal conditions comprising a nucleic acid construct capable of producing a nucleic acid molecule having at least one stem-loop structure wherein said construct comprises:

(i) two first segments, wherein each first segment (a) is complementary to a strand of the HBV nucleic acid sequence or complement thereof and (b) comprises an extendible 3′

end and

(ii) a second segment capable of forming a stem-loop structure after extension of one of said first segments using said HBV nucleic acid sequence or the complement thereof as a template.

7. The composition of claim 6, wherein said nucleic acid construct is capable of producing a nucleic acid molecule comprising a pair of complementary stem-loop structures.

8. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest under isothermal conditions comprising two primers wherein the two 5′

ends of the primers are joined together to form a nucleic acid construct, said construct capable of producing a nucleic acid molecule having at least one stem-loop structure, wherein said construct comprises

(i) two first segments, wherein each first segment is complementary to a strand of said HBV nucleic acid sequence or complement thereof and

(ii) a second segment capable of forming a stem-loop structure after extension of one of said first segments with said HBV nucleic acid sequence or complement thereof as a template.

9. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest under isothermal conditions comprising two primers, wherein the two 5′

ends of the primers are joined together to form a nucleic acid construct, said construct capable of producing a nucleic acid molecule having at least one stem-loop structure, wherein said construct comprises (i) two first segments, wherein each first segment is complementary to a strand of said HBV nucleic acid sequence or complement thereof and (ii) two second segments capable of forming a stem-loop structure after extension of one of said first segments with said HBV nucleic acid sequence or complement thereof as a template.

10. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest under isothermal conditions comprising a primer comprising:

(A)one or more first segments that are substantially complementary to sequences in only one strand of said HBV nucleic acid sequence such that said one or more first segments can bind and be extended using only said strand of said HBV nucleic acid sequence as a template;

(B) one or more second segments that are substantially identical to a portion of said HBV nucleic acid sequence such that said one or more second segments are capable of self-hybridization with sequences created by extension of a first segment of the primer using said HBV nucleic acid sequence as a template, thereby allowing a secondary structure to form that promotes self-priming events, and

(C)one or more third segments that are capable of acting as intrastrand templates and thereby allowing self-extension.

11. The composition of claim 10, wherein said primer has one polarity.

12. The composition of claim 10, wherein said primer has multiple polarities.

13. The composition according to claim 10, wherein said primer has at least two first segments, two second segments and two third segments.

14. The composition according to claim 13, wherein said first segments are substantially identical to each other.

15. The composition according to claim 13, wherein said first segments are substantially dissimilar to each other.

16. The composition according to claim 13, wherein said second segments are substantially identical to each other.

17. The composition according to claim 13, wherein said second segments are substantially dissimilar to each other.

18. The composition according to claim 13, wherein said third segments are substantially identical to each other.

19. The composition according to claim 13, wherein said third segments are substantially dissimilar to each other.

20. A composition useful for the production of two or more copies of a hepatitis B virus (HBV) nucleic acid sequence of interest comprising:

(i) at least one first initial primer comprising two segments;

(A) a first segment (1) being sufficiently complementary to a first portion of said HBV nucleic acid sequence to hybridize thereto, and (2) capable of template dependent extension to produce a first primer extension product, and

(B) a second segment being (1) substantially non-identical and substantially non-complementary to said first segment, and (2) substantially identical to a second portion of said specific nucleic acid sequence, such that said second segment of said first initial primer is capable of hybridizing to said first primer extension product, thereby providing for subsequent binding of a first segment of a second copy of said first initial primer to said first portion of said HBV nucleic acid sequence under isothermal or limited cycling conditions, such that a second copy of said first primer extension is produced and displaces said first primer extension product; and

(ii) at least one second initial primer comprising two regions;

(A) a first region (1) being sufficiently complementary to a first portion of said first primer extension product to hybridize thereto and (2) capable of template-dependent extension to produce a second primer extension product and (B) a second region being (1) substantially nonidentical and substantially non-complementary to said first region, and (2) substantially identical to a second portion of said first primer extension product, such that said second region is capable of hybridizing to the complement of said second portion of said first primer extension product formed by extension of the first region with said first primer extension product as a template, thereby providing for binding of a second copy of a second initial primer to said first portion of said first extension product under isothermal or limited cycling conditions,

wherein the 5′

end of said second segment of said first initial primer is joined to the 5′

end of the second region of the second initial primer.