Nested oligonucleotides containing a hairpin for nucleic acid amplification

Abstract

Templates that are engineered to contain a predetermined sequence and a hairpin structure are provided by a nested oligonucleotide extension reaction. The engineered template allows Single Primer Amplification (SPA) to amplify a target sequence within the engineered template. In particularly useful embodiments, the target sequences from the engineered templates are cloned into expression vehicles to provide a library of polypeptides or proteins, such as, for example, an antibody library.

1. A method of amplifying nucleic acid comprising the steps of:
 a) annealing a primer to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence;  
 b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template adjacent to the location at which the first portion of the primer anneals to the template, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer,  
 c) separating the polynucleotide synthesized in step (b) from the template;  
 d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure;  
 e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and  
 f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.  
 
     
     
       2. A method as in  claim 1  further comprising the step of providing a nucleic acid template by annealing a restriction oligonucleotide to a nucleic acid strand to form a double stranded portion and digesting the nucleic acid strand at the double stranded portion. 
     
     
       3. A method as in  claim 1  wherein the template encodes an immunoglobulin molecule or fragment thereof. 
     
     
       4. A method as in  claim 1  wherein the template is selected from the group consisting of full length or truncated mRNA, DNA and cDNA. 
     
     
       5. A method as in  claim 1  wherein the nucleic acid being amplified includes a target sequence encoding a polypeptide. 
     
     
       6. A method as in  claim 5  wherein the target sequence encodes an immunoglobulin molecule or fragment thereof. 
     
     
       7. A method as in  claim 5  further comprising the step of digesting the extended polynucleotide to isolate the target sequence. 
     
     
       8. A method as in  claim 7  further comprising the step of ligating the isolated target sequence into an expression vector. 
     
     
       9. A method as in  claim 8  further comprising the steps of transforming a host cell with the expression vector and expressing the polypeptide encoded by the target sequence. 
     
     
       10. A method of amplifying nucleic acid comprising the steps of:
 a) annealing a primer and a boundary oligonucleotide to a template nucleic acid sequence, the primer having a first portion which anneals to the template and a second portion of predetermined sequence;  
 b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer;  
 c) separating the polynucleotide synthesized in step (b) from the template;  
 d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide and a second portion having a hairpin structure;  
 e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and  
 f) amplifying the extended polynucleotide using a single primer having the predetermined sequence.  
 
     
     
       11. A method as in  claim 10  further comprising the step of providing a nucleic acid template by generating first strand cDNA from mRNA. 
     
     
       12. A method as in  claim 10  wherein the template is selected from the group consisting of full length or truncated mRNA, DNA and cDNA. 
     
     
       13. A method as in  claim 10  wherein the extended polynucleotide includes a target sequence encoding a polypeptide. 
     
     
       14. A method as in  claim 10  wherein the extended polynucleotide encodes an immunoglobulin molecule or fragment thereof. 
     
     
       15. A method as in  claim 14  wherein the target sequence encodes an immunoglobulin molecule or fragment thereof. 
     
     
       16. A method as in  claim 14  further comprising the step of digesting the extended polynucleotide to isolate the target sequence. 
     
     
       17. A method as in  claim 16  further comprising the step of ligating the isolated target sequence into an expression vector. 
     
     
       18. A method as in  claim 17  further comprising the steps of transforming a host cell with the expression vector and expressing the polypeptide encoded by the target sequence. 
     
     
       19. A method of amplifying nucleic acid comprising the steps of:
 a) annealing an oligo dT primer and a boundary oligonucleotide to an mRNA template;  
 b) synthesizing a polynucleotide that anneals to and is complementary to the portion of the template between the location at which the first portion of the primer anneals to the template and the portion of the template to which the boundary oligonucleotide anneals, the polynucleotide having a first end and a second end, wherein the first end incorporates the primer;  
 c) separating the polynucleotide synthesized in step (b) from the template;  
 d) annealing a nested oligonucleotide to the second end of the polynucleotide synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotide, and a second portion having a hairpin structure;  
 e) extending the polynucleotide synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a poly A terminal portion; and  
 f) amplifying the extended polynucleotide using a single primer.  
 
     
     
       20. A method as in  claim 19  further comprising the step of providing a nucleic acid template by generating first strand cDNA from mRNA. 
     
     
       21. A method as in  claim 19  wherein the template is selected from the group consisting of full length or truncated mRNA, DNA and cDNA. 
     
     
       22. A method as in  claim 19  wherein the extended polynucleotide includes a target sequence encoding a polypeptide. 
     
     
       23. A method as in  claim 19  wherein the extended polynucleotide encodes an immunoglobulin molecule or fragment thereof. 
     
     
       24. A method as in  claim 22  wherein the target sequence encodes an immunoglobulin molecule or fragment thereof. 
     
     
       25. A method as in  claim 22  further comprising the step of digesting the extended polynucleotide to isolate the target sequence. 
     
     
       26. A method as in  claim 25  further comprising the step of ligating the isolated target sequence into an expression vector. 
     
     
       27. A method as in  claim 26  further comprising the steps of transforming a host cell with the expression vector and expressing the polypeptide encoded by the target sequence. 
     
     
       28. A method of amplifying a nucleic acid strand comprising the steps of:
 a) providing a nucleic acid strand having i) a predetermined sequence engineered onto a first end thereof, ii) a sequence complementary to the predetermined sequence, and iii) a hairpin structure therebetween; and  
 b) contacting the engineered nucleic acid strand with a primer containing at least a portion of the predetermined sequence in the presence of a polymerase and nucleotides under conditions suitable for polymerization of the nucleotides, thereby producing a complementary nucleic acid strand.  
 
     
     
       29. A method as in  claim 28  further comprising the steps of:
 digesting the complementary nucleic acid strand to isolate a target nucleic acid sequence contained therein;  
 ligating the target nucleic acid sequence into an expression vector;  
 transforming a host organism with the expression vector; and  
 
       expressing a polypeptide or protein encoded by the target sequence. 
     
     
       30. A method of amplifying a family of related nucleic acid sequences to build a complex library of polypeptides encoded by the sequences, the method comprising:
 a) annealing a primer to a family of related nucleic acid sequence templates, the primer having a first portion which anneals to the templates and a second portion of predetermined sequence;  
 b) synthesizing polynucleotides that anneal to and are complementary to the portion of the templates adjacent to the location at which the first portion of the primer anneals to the templates, the polynucleotides having a first end and a second end, wherein the first end incorporates the primer;  
 c) separating the polynucleotides synthesized in step (b) from the templates;  
 d) annealing a nested oligonucleotide to the second end of the polynucleotides synthesized in step (b), the nested oligonucleotide having a first portion that anneals to the second end of the polynucleotides, and a second portion having a hairpin structure;  
 e) extending the polynucleotides synthesized in step (b) to provide an extended polynucleotide comprising a portion that is complementary to the hairpin structure and a terminal portion that is complementary to the predetermined sequence; and  
 f) amplifying the extended polynucleotides using a single primer having the predetermined sequence.  
 
     
     
       31. A method as in  claim 1 , wherein steps a), b) and c) are repeated from 15 to 25 times prior to annealing the nested oligonucleotide. 
     
     
       32. A method as in  claim 10 , wherein steps a), b) and c) are repeated from 15 to 25 times prior to annealing the nested oligonucleotide. 
     
     
       33. A method as in  claim 19 , wherein steps a), b) and c) are repeated from 15 to 25 times prior to annealing the nested oligonucleotide. 
     
     
       34. A method as in  claim 30 , wherein steps a), b) and c) are repeated from 15 to 25 times prior to annealing the nested oligonucleotide. 
     
     
       35. A method as in  claim 1  wherein the first end of the polynucleotide is the 5′ end. 
     
     
       36. A method as in  claim 1  wherein the first end of the polynucleotide is the 5′ end. 
     
     
       37. A method as in  claim 19  wherein the first end of the polynucleotide is the 5′ end. 
     
     
       38. A method as in  claim 1  wherein the first end of the nucleic acid strand is the 5′ end.