Universal-tagged oligonucleotide primers and methods of use

Abstract

The present invention relates to universal-tagged oligonucleotide primers, and to methods of using the primers for amplifying the genome.

1 . A method for amplifying target DNA by polymerase chain reaction (PCR) comprising: 
 preparing a mixture of (1) said target DNA, comprising multiple DNA sequences, (2) a set of single-stranded oligonucleotide primers, each primer comprising (i) a 3′ specific region, (ii) a random region, and (iii) a 5′ universal region, wherein said universal region serves as a first universal priming site (U1) for further PCR amplification with a complementary primer, (3) a DNA polymerase, and (4) multiple deoxynucleoside triphosphates (dNTPs) under conditions such that said PCR primers anneal to target DNA; and    preparing a copy of said multiple DNA sequences in said target DNA, whereby an amplified product is formed incorporating said first universal priming site (U1).    
     
     
         2 . The method of  claim 1  wherein said target DNA is genomic DNA (gDNA).  
     
     
         3 . The method of  claim 1  wherein said target DNA is complementary DNA (cDNA).  
     
     
         4 . The method of  claim 2  wherein said gDNA is human.  
     
     
         5 . The method of  claim 4  wherein at least a fraction of the whole human genome is amplified from said gDNA.  
     
     
         6 . The method of  claim 5  wherein said 3′ specific region is designed to bind to a genomic sequence occurring in the human genome with a frequency of between about 0.01% and 2.0%.  
     
     
         7 . The method of  claim 1  wherein said (i) 3′ specific region is between about 4 and 12 bases in length.  
     
     
         8 . The method of  claim 7  wherein said 3′ specific region is 5 bases in length.  
     
     
         9 . The method of  claim 8  wherein the sequence of said 3′ specific region is selected from the group consisting of: AAAAA; AAAAT; AAATA; TAAAA; AGAAA; ATAAA; AAATT; TTAAA; GAAAA; AATAA; CAAAA; AAGAA; AAAGA; AAAAG; AAATG; AAACA; ACAAA; ATTTA; TGAAA; and ATATA; AATAT; AAAAC; AGAGA; AATTA; AATTA; AGGAA; AACAA; CCCAG; TCAAA; ATTTC; ATAAT; ACACA; CAGAA; AGAAT; GAGAA; GGAAA; TCCCA; ATTAA; ATGAA; AAAGT; AATGA; TTCAA; ATTTG; TCTCA; ACAGA; AAACT; CCAGG; AGAAG; and TATAA.  
     
     
         10 . The method of  claim 1  wherein the set of oligonucleotide primers used are determined according to bioinformatic prediction of expected products using in silico e-PCR.  
     
     
         11 . The method of  claim 1 , wherein the primer consists of (i) a 3′ specific region, (ii) a random region, and (iii) a 5′ universal region, wherein said universal region serves as a first universal priming site (U1) for further PCR amplification with a complementary primer.  
     
     
         12 . A method for amplifying target DNA by polymerase chain reaction (PCR) comprising: 
 providing a length of a PCR amplified product that is primed at a particular specified site on a target DNA;    preparing a mixture, of (1) said target DNA, comprising multiple DNA sequences, (2) a set of oligonucleotide primers, each primer comprising (i) a 3′ specific region, wherein a sequence of said 3′ specific region is selected to hybridize to the specified site on the target DNA, wherein said 3′ specific region includes at least one locked nucleic acid (LNA), and wherein said specified site on the target DNA is chosen according to the length of the PCR amplified product that is primed at the specified site on the target DNA and (ii) a 5′ universal region, wherein said universal region is selected to meet a requirement of a PCR primer and serves as a first universal priming site (U1) for further PCR amplification with a complementary primer, wherein the 5′ universal region, can serve as an entire first universal priming site, independent of the 3′ specific region, (3) a DNA polymerase, and (4) multiple deoxynucleoside triphosphates (dNTPs) under conditions such that said PCR primers anneal to target DNA; and    performing a polymerase chain reaction (PCR) to amplify the target DNA sequences in said target DNA, whereby a PCR amplified product is formed incorporating said first universal priming site (U1).    
     
     
         13 . The method of  claim 12 , wherein the primer further comprises a random region of nucleic acids located between the specific region and the universal region.  
     
     
         14 . The method of  claim 12 , wherein there are 5 or fewer LNAs in the specific region.  
     
     
         15 . The method of  claim 12 , wherein there are 3 or fewer LNAs in the specific region.  
     
     
         16 . The method of  claim 12 , wherein the specific region is at least 9 nucleic acids long.  
     
     
         17 . An oligonucleotide primer for polymerase chain reaction (PCR), comprising (1) a distinct 3′ specific region of between about 4 and 12 bases in length, wherein said 3′ specific region hybridizes to a single specified site on a target, wherein the 3′ specific region includes at least one locked nucleic acid (LNA), and wherein the specified site is configured so that a PCR product initiated at the specified site on the target by the 3′ specific region will result in products with an average length of between 74 and 523 bases and (2) a distinct 5′ universal region of about 12 and 35 bases in length, wherein the universal region does not bind to an initial target DNA when the 3′ specific region binds to the initial target DNA, and wherein the 5′ universal region hybridizes to a second primer so as to allow priming at the 5′ universal region.  
     
     
         18 . The oligonucleotide primer of  claim 17 , further comprising a distinct random region of between about 2 and 15 bases in length, wherein the random region is located between the 3′ specific region and the 5′ universal region.  
     
     
         19 . The oligonucleotide primer of  claim 17 , wherein there are 3 or fewer LNAs in the specific region.  
     
     
         20 . The oligonucleotide primer of  claim 17 , wherein the specific region is at least 9 nucleic acids long.