Nucleic acid extraction and amplification controls and methods of use thereof

Abstract

Nucleic acid reagents and corresponding methods of using the same for monitoring and evaluating nucleic acid extraction and amplification reactions.

1 . An array for nucleic acid amplification, comprising:
 a support containing a plurality of reaction sites located within the support or upon the support; each of the plurality of reaction sites containing:
 (i) a control nucleic acid molecule containing a plurality of different target sequences, 
 (ii) an amplification primer pair configured to amplify a corresponding target sequence, and 
 (iii) a detectably labeled probe configured to hybridize to a nucleic acid sequence generated by extension of at least one of the amplification primers of the pair. 
   
     
     
         2 . The array of  claim 1 , wherein at least two of the different target sequences include a sequence selected from the group consisting of the sequences listed in Table 2 or its complement. 
     
     
         3 . The array of  claim 1 , wherein the control nucleic acid molecule is a plasmid. 
     
     
         4 . The array of 1, wherein at least one of the reaction sites includes an amplification product. 
     
     
         5 . The array of  claim 1 , wherein at least two of the reaction sites each contains a pair of amplification primers configured to amplify a different corresponding target sequence. 
     
     
         6 . The array of  claim 1 , wherein the detectably labeled probe of at least one reaction site contains a fluorescent label at its 5′ end and a quencher at its 3′ end. 
     
     
         7 . The array of  claim 1 , wherein the support is selected from a multi-well plate, a microfluidic card, and a plate containing a plurality of through-hole reaction sites. 
     
     
         8 . The array of  claim 1 , wherein the plurality of reaction sites further include a polymerase and nucleotides. 
     
     
         9 . The array of  claim 1 , wherein at least two of the different target sequences comprise at least a 20 nucleotide portion of a gene selected from Table 3 or its corresponding cDNA. 
     
     
         10 . The array of  claim 1 , wherein each reaction site contains a pair of amplification primers and a probe configured to amplify a sequence which includes a sequence selected from the group consisting of the sequences listed in Table 2 or its complement. 
     
     
         11 . The array of  claim 1 , wherein the control nucleic acid molecule contains at least ten different target sequences. 
     
     
         12 . The array of  claim 11 , wherein the control nucleic acid molecule contains at least twenty different target sequences. 
     
     
         13 . The array of  claim 12 , wherein the control nucleic acid molecule contains at least thirty different target sequences. 
     
     
         14 . The array of  claim 13 , wherein the control nucleic acid molecule contains about twenty to about fifty different target sequences. 
     
     
         15 . The array of  claim 3 , wherein the plasmid is linear. 
     
     
         16 . The array of  claim 1 , wherein the support includes between 10 and 10,000 reaction sites containing different amplification products. 
     
     
         17 . The array of  claim 1 , wherein the detectably labeled probe of at least one reaction site is configured to undergo cleavage by a polymerase in a 5′ nuclease assay. 
     
     
         18 . The array of  claim 1 , wherein the detectably labeled probe further contains a minor groove binder moiety.