Single-step dna preparation for polymerase chain reaction using magnetic chitosan microparticles

Abstract

The present invention relates to a method for concentrating a biological sample containing nucleic acids by using magnetic chitosan microparticles and subsequently performing a PCR reaction on the nucleic acids captured on the microparticles. The chitosan microparticles added to the biological sample at a PCR compatible pH are mechanically agitated to provide for cell lysis and simultaneous DNA capture, and then serve as a solid support for the nucleic acid template during the PCR reaction. As the chitosan microparticles are utilized for lysis and the nucleic acids do not need to be removed from the microparticles before PCR, the ease of the sample preparation procedure is dramatically improved.

1 . A method of purifying nucleic acids from a biological sample comprising one or more cells, the method comprising the steps of:
 adding microparticles to the biological sample having a PCR amplification compatible pH;   lysing the one or more cells in the biological sample by mechanically agitating the biological sample; and   capturing released nucleic acids on the microparticles.   
     
     
         2 . The method of  claim 1 , wherein the microparticles are fabricated from chitosan. 
     
     
         3 . The method of  claim 1 , wherein the microparticles are magnetic microparticles. 
     
     
         4 . The method of  claim 2 , wherein the chitosan microparticles are fabricated by creating chitosan droplets in oil and then crosslinking the droplets into microparticles. 
     
     
         5 . The method of  claim 4 , wherein crosslinking the droplets into microparticles goes into a core of microparticles over time. 
     
     
         6 . The method of  claim 1 , further comprising embedding magnetic nanoparticles within individual microparticles. 
     
     
         7 . The method of  claim 6 , wherein the magnetic nanoparticles are iron nanoparticles measuring 20-40 nm in diameter. 
     
     
         8 . The method of  claim 1 , further comprising, following purification, amplifying the nucleic acids from a surface of the magnetic microparticles, wherein the purification is performed with the same buffer as the amplification. 
     
     
         9 . The method of  claim 1 , wherein the lysis is performed by mechanically agitating the biological sample including vortexing the biological sample with the magnetic microparticles. 
     
     
         10 . The method of  claim 1 , wherein the step of lysing the one or more cells in the biological sample includes heating the biological sample and vortexing the biological sample. 
     
     
         11 . The method of  claim 1 , further comprising separating and washing microparticles with captured nucleic acids. 
     
     
         12 . The method of  claim 2 , wherein the chitosan microparticles provide for cell lysis and simultaneous DNA capture, and then serve as a solid support for a DNA template during a PCR amplification reaction. 
     
     
         13 . The method of  claim 2 , wherein interior of the chitosan microparticle, beneath a charge-switchable shell, remains positively charged at high pH. 
     
     
         14 . The method of  claim 1 , wherein nucleic acids are selected from the group consisting of: plasmids, bacterial DNA, and human genomic DNA. 
     
     
         15 . A microfluidic system for amplifying nucleic acids from a biological sample, the system comprising:
 a biological sample combined with microparticles;   a lysis unit configured to mechanically agitate the biological sample combined with microparticles to lyse cells in the biological sample, wherein nucleic acids released from the cells are captured on the magnetic microparticles;   a purification unit configured to purify the nucleic acids released in the biological sample as result of lysis; and   an amplification unit configured to accept the purified nucleic acid to perform amplification directly on the captured nucleic acids.   
     
     
         16 . The system of  claim 15 , wherein the microparticles are fabricated from chitosan. 
     
     
         17 . The system of  claim 15 , wherein the microparticles are magnetic microparticles. 
     
     
         18 . The system of  claim 16 , wherein the chitosan microparticles are fabricated by creating chitosan droplets in oil and then crosslinking the droplets into microparticles. 
     
     
         19 . The system of  claim 18 , wherein crosslinking the droplets into microparticles goes into a core of microparticles over time. 
     
     
         20 . The system of  claim 15 , wherein nanoparticles are embedded within individual microparticles. 
     
     
         21 . The system of  claim 20 , wherein the nanoparticles are iron nanoparticles measuring 20-40 nm in diameter. 
     
     
         22 . The system of  claim 15 , wherein the nucleic acids are amplified from a surface of the magnetic microparticles, the purification being performed with the same buffer as the amplification. 
     
     
         23 . The system of  claim 15 , wherein the lysis unit is configured to mechanically agitate the biological sample by vortexing the biological sample with the magnetic microparticles. 
     
     
         24 . The system of  claim 15 , wherein lysing the one or more cells in the biological sample includes heating the biological sample prior to vortexing the biological sample having microparticles therein. 
     
     
         25 . The system of  claim 15 , wherein the purification unit is configured to separate and wash microparticles with captured nucleic acids. 
     
     
         26 . The system of  claim 16 , wherein the chitosan microparticles provide for cell lysis and simultaneous DNA capture, and then serve as a solid support for a DNA template during a PCR amplification reaction. 
     
     
         27 . The system of  claim 16 , wherein an interior of the chitosan microparticle, beneath a charge-switchable shell, remains positively charged at high pH. 
     
     
         28 . The system of  claim 15 , wherein the nucleic acids are selected from the group consisting of: plasmids, bacterial DNA, and human genomic DNA.