Method of producing a DNA library using positional amplification

Abstract

The disclosed invention relates to general and specific methods to use the Primer Extension/Nick Translation (PENT) reaction to create an amplifiable DNA strand, called a PENTAmer. A PENTAmers can be made for the purpose of amplifying a controlled length of DNA located at a controlled position within a DNA molecule, a process referred to as Positional Amplification by Nick Translation (PANT). In contrast to PCR, which amplifies DNA between two specific sequences, PANT can amplify DNA between two specific positions. PENTAmers can be created to amplify-very large regions of DNA (up to 500,000 bp) as random mixtures (unordered positional libraries), or as molecules sorted according to position (ordered positional libraries). PANT is fast and economical, because PENTAmer preparation can be multiplexed. A single PENTAmer preparation can include very complex mixtures of DNA such as hundreds of large-insert clones, complete genomes, or cDNA libraries. Subsequent PCR amplification of the preparation using a single specific primer can positionally amplify contiguous regions along a specific clone, along a specific genomic region, or along a specific expressed sequence.

1 - 189 . (canceled)  
     
     
         190 . A kit comprising amplifiable DNA, wherein said DNA is prepared by the method comprising: 
 a) obtaining a DNA sample comprising DNA molecules having regions to be amplified;    b) attaching upstream adaptor molecules to ends of DNA molecules of the sample to provide a nick translation initiation site;    c) subjecting the DNA molecules to nick translation comprising DNA polymerization and 5′-3′ exonuclease activity to produce nick translate molecules; and    d) attaching downstream adaptor molecules to the nick translate molecules to produce adaptor attached nick translate molecules.    
     
     
         191 . The kit of  claim 190 , wherein said DNA is genomic DNA.  
     
     
         192 . The kit of  claim 191 , wherein said genomic DNA is isolated from a prokaryote.  
     
     
         193 . The kit of  claim 191 , wherein said genomic DNA is isolated from a eukaryltic eukaryote.  
     
     
         194 . The kit of  claim 191 , wherein said genomic DNA is isolated from an animal.  
     
     
         195 . The kit of  claim 194 , wherein said animal is selected from the group consisting of human, feline, canine, bovine, equine, porcine, caprine, murine, lupine, ranine, piscine and simian  
     
     
         196 . The kit of  claim 191 , wherein said genomic DNA is isolated from a plant.  
     
     
         197 . The kit of  claim 196 , wherein said plant is a dicotyledonous plant.  
     
     
         198 . The kit of  claim 197 , wherein said dicotyledonous plant is selected from the group consisting of tobacco, tomato, potato, sugar beet, pea, carrot, cauliflower, broccoli, soybean, canola, sunflower, alfalfa, cotton and  Arabidopsis.    
     
     
         199 . The kit of  claim 195 , wherein said DNA is isolated from a monocotyledonous plant.  
     
     
         200 . The kit of  claim 199 , wherein said monocotyledonous plant is selected from the group consisting of wheat, maize, rye, rice, turfgrass, oat, barley, sorghum, millet, and sugarcane.  
     
     
         201 - 272 . (canceled)