Selective restriction fragment amplification: fingerprinting

Abstract

The invention relates to a process for the controlled amplification of at least one part of a starting DNA containing a plurality of restriction sites for a determined specific restriction endonuclease, and of which at least part of its nucleic acid is unknown. This technology can be applied to human, animal or plant DNA fingerprinting, to identify restriction fragment length polymorphisms. Also encompassed by the inventive technology are kits for the application of the process.

1 . A method of amplifying a restriction fragment from at least one source of DNA comprising:
 (a) digesting said source of DNA with at least one restriction endonuclease that cleaves the DNA at its recognized target site to produce restriction fragments;   (b) ligating in molar excess at least one adaptor to the ends of at least one restriction fragment, wherein each adaptor comprises (i) a first end that is designed to ligate to said restriction fragment; (ii) a second end that is designed so that it cannot ligate to said restriction fragment, and (iii) situated between said first and second ends unique nucleotide sequences at specific positions; and   (c) amplifying said tagged restriction fragment with two primers each of which comprises a nucleotide sequence complementary to a unique nucleotide sequence in said adaptor to produce amplified restriction fragments from said source of DNA.   
     
     
         2 . The method of  claim 1 , wherein said restriction fragment comprises a single-stranded extension and said adaptor comprises a single-stranded extension which is complementary to said single-stranded extension of said restriction fragment. 
     
     
         3 . The method of  claim 1 , where an end of said restriction fragment is flush, and wherein an end of said adaptor is flush, and wherein the flush ends of said restriction fragment and said adaptor are ligated together. 
     
     
         4 . The method of  claim 1 , wherein said amplified restriction fragment is used in a method selected from the group consisting of forensic typing, microbial identification, varietal identification, pedigree analysis and screening of genetic markers linked to genetic traits. 
     
     
         5 . The method of  claim 1 , wherein said amplified restriction fragment is used in a method selection from the group consisting of screening of genetically inherited disease in humans, monitoring the inheritance of agronomic traits in plant and animal breeding and detection of infectious agents in diseases. 
     
     
         6 . The method of  claim 1 , wherein said source of DNA is selected from the group consisting of plants, animal and microbes. 
     
     
         7 . The method of  claim 1 , wherein said source of DNA correlates to a unique sequence in said adaptor. 
     
     
         8 . A method of preparing a nucleic acid for sequencing from a source of DNA comprising:
 (a) digesting said source of DNA with at least one restriction endonuclease that cleaves the DNA at its recognized target site to produce restriction fragments;   (b) ligating molar excess at least one adaptor to the ends of at least one restriction fragment, wherein each adaptor comprises (i) a first end that is designed to ligate to said restriction fragment; (ii) a second end that is designed so that it cannot ligate to said restriction fragment, and (iii) situated between said first and second ends unique nucleotide sequences at specific positions;   (c) amplifying said tagged restriction fragment with two primers each of which comprises a nucleotide sequence complementary to a unique nucleotide sequence in said adaptor to produce amplified restriction fragments from said source of DNA, and   (d) sequencing said tagged restriction fragment.   
     
     
         9 . The method of  claim 8 , wherein said source of DNA is selected from the group consisting of plants, animal and microbes. 
     
     
         10 . The method of  claim 8 , wherein said source of DNA correlates to a unique sequence in said adaptor. 
     
     
         11 . The method of  claim 8 , wherein said amplified restriction fragment is used in a method selected from the group consisting of forensic typing, microbial identification, varietal identification, pedigree analysis and screening of genetic markers linked to genetic traits. 
     
     
         12 . The method of  claim 8 , wherein said amplified restriction fragment is used in a method selection from the group consisting of screening of genetically inherited disease in humans, monitoring the inheritance of agronomic traits in plant and animal breeding and detection of infectious agents in diseases. 
     
     
         13 . A method of identifying an amplified restriction fragment from a source of DNA comprising:
 (a) digesting said source of DNA with at least one restriction endonuclease that cleaves the DNA at its recognized target site to produce restriction fragments;   (b) ligating in molar excess at least one adaptor to the ends of at least one restriction fragment, wherein each adaptor comprises (i) a first end that is designed to ligate to said restriction fragment; (ii) a second end that is designed so that it cannot ligate to said restriction fragment, and (iii) situated between said first and second ends unique nucleotide sequences at specific positions;   (c) amplifying said tagged restriction fragment with two primers each of which comprises a nucleotide sequence complementary to at least a portion of said adaptor to produce amplified restriction fragments from said source of DNA; and   (d) identifying said amplified restriction fragments with a labelled hydrization probe comprising a nucleotide sequence complementary to a unique nucleotide sequence in said amplified restriction fragments.   
     
     
         14 . The method of  claim 13 , wherein said restriction fragment comprises a single-stranded extension and said adaptor comprises a single-stranded extension which is complementary to said single-stranded extension of said restriction fragment. 
     
     
         15 . The method of  claim 13 , where an end of said restriction fragment is flush, and wherein and end of said adaptor is flush, and wherein the flush ends of said restriction fragment and said adaptor are ligated together. 
     
     
         16 . The method of  claim 13 , wherein said amplified restriction fragment is used in a method selected from the group consisting of forensic typing, microbial identification, varietal identification, pedigree analysis and screening of genetic markers linked to genetic traits. 
     
     
         17 . The method of  claim 13 , wherein said amplified restriction fragment is used in a method selection from the group consisting of screening of genetically inherited disease in humans, monitoring the inheritance of agronomic traits in plant and animal breeding and detection of infectious agents in diseases. 
     
     
         18 . A method of providing a fingerprint of a genome comprising:
 (a) digesting a source of DNA with at least one restriction endonuclease that cleaves the DNA at its recognized target site to produce restriction fragments;   (b) ligating at least one adaptor to the ends of restriction fragments, wherein each adaptor comprises (i) a first end that is designed to ligate to said restriction fragment; (ii) a second end that is designed so that it cannot ligate to said restriction fragment, and (iii) situated between said first and second ends unique nucleotide sequences at specific positions;   (c) amplifying said tagged restriction fragments with primers each of which comprises a nucleotide sequence complementary to a unique nucleotide sequence in said adaptor to produce amplified restriction fragments from said source of DNA, and   (d) establishing an amplified restriction fragment pattern that provides a fingerprint of the genome.   
     
     
         19 . A method of linking an amplified restriction fragment to a specific trait comprising:
 (a) digesting a source of DNA from at least two different individuals, wherein said individuals exhibit differences in the specific trait, with at least one restriction endonuclease that cleaves the DNA at its recognized target site to produce restriction fragments;   (b) ligating at least one adaptor to the ends of restriction fragments, wherein each adaptor comprises (i) a first end that is designed to ligate to said restriction fragment; (ii) a second end that is designed so that it cannot ligate to said restriction fragment, and (iii) situated between said first and second ends unique nucleotide sequences at specific positions;   (c) amplifying said tagged restriction fragments with primers each of which comprises a nucleotide sequence complementary to a unique nucleotide sequence in said adaptor to produce amplified restriction fragments from said source of DNA, and   (d) correlating the inheritance of one or more amplified restriction fragments with the phenotype exhibited by said specific trait.