Method of genome-wide nucleic acid fingerprinting of functional regions

Abstract

A method of specifically amplifying desired regions of nucleic acid from a sample is provided. The method uses a plurality of first and second PCR primers, each having a region of fixed nucleotide sequence identical or complementary to a consensus sequence of interest and a region of randomized nucleotide sequence located 5′ to, 3′ to, anywhere within, or flanking the region of fixed nucleotide sequence; and then amplifying the nucleic acid present in the sample via PCR using the plurality of first and second PCR primers; whereby a subset of the first primers binds to the consensus sequence of interest wherever it occurs in the sample, and a subset of the second primers binds to the sample at locations removed from the first primers such that DNA regions flanked by the first primer and the second primer are specifically amplified.

1 .- 14 . (canceled) 
     
     
         15 . A plurality of primers, comprising, in combination:
 a plurality of PCR primers having a region of fixed nucleotide sequence identical or complementary to a target sequence of interest and a region of randomized nucleotide sequence located 5′ to, 3′ to, or flanking the region of fixed nucleotide sequence, wherein all possible nucleotide sequences within the region of randomized nucleotide sequence are contained within the plurality of PCR primers.   
     
     
         16 . The plurality of primers of claim  1 , wherein in the plurality of PCR primers, each primer comprises a region of fixed nucleotide sequence complementary to a target sequence selected from the group consisting of a promoter sequence, a 3′ splice sequence, a 5′ splice sequence, an Ala repeat, a tandem repeat, poly-A site, a lariat signal, a microsatellite sequence, and a homeobox sequence, or any sequence that is repeated more than once within a given template nucleic acid. 
     
     
         17 . The plurality of primers of claim  1 , wherein the plurality of primers has a G+C content greater than 50%. 
     
     
         18 . The plurality of primers of claim  1 , wherein the plurality of primers has a G+C content less than 50%. 
     
     
         19 . The plurality of primers of claim  1 , wherein the plurality of primers has a G+C content of about 50%. 
     
     
         20 . The plurality of primers of claim  1 , wherein the plurality of primers has an overall length of from about 10 nucleotides to about 36 nucleotides. 
     
     
         21 . A plurality of primers, comprising, in combination:
 a plurality of first PCR primers having a region of fixed nucleotide sequence identical or complementary to a consensus sequence of interest and a region of randomized nucleotide sequence located 5′ to, 3′ to, or flanking the region of fixed nucleotide sequence; and   a plurality of second PCR primers having a region of arbitrary, yet fixed nucleotide sequence and a region of randomized nucleotide sequence located 5′ to, 3′ to, or flanking the region of fixed nucleotide sequence, wherein all possible nucleotide sequences within the region of randomized nucleotide sequence are contained within the plurality of second PCR primers.   
     
     
         22 . The plurality of primers of  claim 21 , wherein in the plurality of first PCR primers, each first primer comprises a region of fixed nucleotide sequence complementary to a consensus sequence selected from the group consisting of a promoter sequence, a 3′ splice sequence, a 5′ splice sequence, an Ala repeat, a tandem repeat, poly-A site, a lariat signal, a microsatellite sequence, and a homeobox sequence. 
     
     
         23 . The plurality of primers of  claim 21 , wherein the plurality of first primers has a G+C content greater than 50%. 
     
     
         24 . The plurality of primers of  claim 21 , wherein the plurality of first primers has a G+C content less than 50%. 
     
     
         25 . The plurality of primers of  claim 21 , wherein the plurality of first primers has a G+C content of about 50%. 
     
     
         26 . The plurality of primers of  claim 21 , wherein the plurality of second primers has a G+C content greater than 50%. 
     
     
         27 . The plurality of primers of  claim 21 , wherein the plurality of second primers has a G+C content less than 50%. 
     
     
         28 . The plurality of primers of  claim 21 , wherein the plurality of first primers has a G+C content of about 50%. 
     
     
         29 . The plurality of primers of  claim 21 , wherein the plurality of first primers has an overall length of from about 10 nucleotides to about 36 nucleotides, and the plurality of second primers has an overall length of from about 10 nucleotides to about 36 nucleotides. 
     
     
         30 . A plurality of primers, comprising, in combination:
 a plurality of first PCR primers, each first primer having:
 (i) a first region of fixed nucleotide sequence dimensioned and configured to hybridize under stringent conditions to a first target sequence of interest, 
 (ii) a region of randomized nucleotide sequence located 5′ to, 3′ to, or flanking the region of fixed nucleotide sequence, and 
 (iii) a second region of fixed nucleotide sequence located at a 5′-terminus or a 3′ terminus of the first primer, 
   wherein all possible nucleotide sequences within the region of randomized nucleotide sequence are contained within the plurality of first PCR primers;   a plurality of second PCR primers, each second primer having:
 (i) a region of fixed nucleotide sequence dimensioned and configured to hybridize under stringent conditions to a second target sequence of interest, and 
 (ii) a region of randomized nucleotide sequence located 5′ to, 3′ to, or flanking the region of fixed nucleotide sequence, 
   wherein all possible nucleotide sequences within the region of randomized nucleotide sequence are contained within the plurality of second PCR primers   
     
     
         31 . The plurality of primers of  claim 30 , wherein in the plurality of first PCR primers, each primer has a first region of fixed nucleotide sequence dimensioned and configured to hybridize under stringent conditions to a first consensus sequence selected from the group consisting of a promoter sequence, a 3′ splice sequence, a 5′ splice sequence, an Alu repeat, a tandem repeat, poly-A site, a lariat signal, a microsatellite sequence, and a homeobox sequence, or to a nucleotide sequence that is repeated many times within the given template DNA/RNA; and wherein in the plurality of second PCR primers, each primer has a region of fixed nucleotide sequence dimensioned and configured to hybridize under stringent conditions to a second consensus sequence selected from the group consisting of a promoter sequence, a 3′ splice sequence, a 5′ splice sequence, an Alu repeat, a tandem repeat, poly-A site, a lariat signal, a microsatellite sequence, and a homeobox sequence, or to a nucleotide sequence that is repeated many times within the given template DNA/RNA. 
     
     
         32 . The plurality of primers of  claim 30 , wherein the plurality of first primers has an overall length of from about 10 nucleotides to about 36 nucleotides, and the plurality of second primers has an overall length of from about 10 nucleotides to about 36 nucleotides. 
     
     
         33 . The plurality of primers of  claim 30 , wherein the plurality of primers is synthesized by chemical or enzymatic means.