Method of nucleic acid amplification

Abstract

A nucleic acid molecule can be annealed to an appropriate immobilized primer. The primer can then be extended and the molecule and the primer can be separated from one another. The extended primer can then be annealed to another immobilized primer and the other primer can be extended. Both extended primers can then be separated from one another and can be used to provide further extended primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. These can be used for many different purposes, including sequencing, screening, diagnosis, in situ nucleic acid synthesis, monitoring gene expression, nucleic acid fingerprinting, etc.

1 . (canceled) 
     
     
         2 . A multiplex method for determining the presence of one or more target polynucleotide sequences across a plurality of samples, comprising:
 amplifying and tagging target polynucleotides by PCR in each of said plurality of samples with an amplification primer comprising a high throughput sequencing adaptor, a sample-specific tag sequence, and a priming sequence to amplify said target polynucleotide sequence(s);   combining the amplified polynucleotides and sequencing the polynucleotide pool in high throughput, so as to determine the sequence of at least 100 tagged polynucleotides for each of said samples;   assigning the nucleotide sequences to the originating samples by the nucleotide sequence of the sample-specific tag, thereby determining the presence of the target polynucleotide sequence(s) across the samples.   
     
     
         3 . The method of  claim 2 , wherein the samples are diagnostic. 
     
     
         4 . The method of  claim 2 , wherein the samples are biological samples. 
     
     
         5 . The method of  claim 2 , wherein the target polynucleotides are genomic DNA or mitochondrial DNA. 
     
     
         6 . The method of  claim 2 , wherein the target polynucleotides are cDNA. 
     
     
         7 . The method of  claim 2 , wherein the number of samples is over 10. 
     
     
         8 . The method of  claim 2 , wherein the number of samples is over 100. 
     
     
         9 . The method of  claim 2 , wherein the priming sequence is the same across samples. 
     
     
         10 . The method of  claim 2 , wherein the sequencing adaptor immobilizes the individual polynucleotides for clonal amplification and sequencing. 
     
     
         11 . The method of  claim 10 , wherein the sequencing is sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-hybridization. 
     
     
         12 . The method of  claim 2 , wherein the PCR amplifications employ a forward and reverse primer that both comprise the high throughput sequencing adaptor and the sample-specific tag. 
     
     
         13 . A method for simultaneously determining the sequence of one or more sequences of interest in a plurality of samples, comprising:
 amplifying and tagging polynucleotide sequences of interest by PCR in each of said plurality of samples with an amplification primer comprising a sequence complementary to a sequencing primer for high throughput sequencing, a tag sequence which is different for each sample, and primer sequences surrounding the specific sequences of interest to amplify said specific sequences of interest;   sequencing the amplified polynucleotide sequences of interest from the plurality of samples in high throughput, so as to determine the sequence of over 100 different sequences from the plurality of samples; and   identifying the origin of the amplified polynucleotide sequences of interest using the tag sequence which is different for each sample.   
     
     
         14 . The method of  claim 13 , wherein the over 100 different sequences are from each of the plurality of samples. 
     
     
         15 . The method of  claim 13 , wherein the method is for diagnosis. 
     
     
         16 . The method of  claim 13 , wherein the samples are biological samples. 
     
     
         17 . The method of  claim 13 , wherein the polynucleotide sequences of interest are genomic DNA or mitochondrial DNA. 
     
     
         18 . The method of  claim 13 , wherein the polynucleotide sequences of interest are cDNA. 
     
     
         19 . The method of  claim 13 , wherein the number of samples is over 10. 
     
     
         20 . The method of  claim 13 , wherein the number of samples is over 100. 
     
     
         21 . The method of  claim 13 , wherein the primer sequences surrounding the specific sequences of interest to amplify said specific sequences of interest is the same across samples. 
     
     
         22 . The method of  claim 13 , wherein the sequence complementary to a sequencing primer for high throughput sequencing immobilizes the individual polynucleotides for clonal amplification and sequencing. 
     
     
         23 . The method of  claim 22 , wherein the sequencing is sequencing-by-synthesis, sequencing-by-ligation, or sequencing-by-hybridization. 
     
     
         24 . The method of  claim 13 , wherein the PCR amplifications employ a forward and reverse primer that both comprise the sequence complementary to a sequencing primer for high throughput sequencing and the tag sequence which is different for each sample. 
     
     
         25 . The method of  claim 13 , wherein over 1000 different sequences from the plurality of samples are determined.