Direct quantification of unprocessed nucleic acid samples

Abstract

A workflow for direct qPCR quantification of unprocessed forensic casework samples is disclosed herein. 13pg of DNA has been detected by direct amplification from a paper substrate. Direct qPCR quantification of unprocessed forensic casework samples and direct STR amplification of unprocessed forensic casework samples collected on the same PE-swab will greatly increase forensic laboratory's efficiency and capability.

1 . A method comprising contacting one or more surfaces with a paper in the collection of evidence for a criminal investigation, depositing the paper into a vessel, performing a real-time polymerase chain reaction (rtPCR) within the vessel and detecting the level of fluorescence emitted from the vessel during rtPCR, wherein the level of fluorescence is detected by a charge-coupled device while the paper is in the vessel. 
     
     
         2 . The method of  claim 1 , wherein one or more surfaces possess a fingerprint. 
     
     
         3 . The method of  claim 1 , wherein the annealing and extension temperatures used in the rtPCR are the same. 
     
     
         4 . The method of  claim 1 , wherein the rtPCR is multiplex rtPCR. 
     
     
         5 . The method of  claim 1 , wherein the fluorescent detection comprising use of a fluorescent reporter probe selected from SYBR green, TaqMan probe, Molecular Beacon, Scorpion Primer, Sunrise Primer, and Eclipse Probe. 
     
     
         6 . The method of  claim 4 , wherein the fluorescent reporter probe is coupled with a quencher. 
     
     
         7 . The method of  claim 1 , wherein the area of the paper is equal to or less than the area of a circle with a diameter of 3 mm. 
     
     
         8 . The method of  claim 1 , wherein the area of the paper is equal to or less than the area of a circle with a diameter of 2 mm. 
     
     
         9 . The method of  claim 1 , wherein the area of the paper is equal to or less than the area of a circle with a diameter of 1 mm.