METHODS FOR PCR SAMPLE PREPARATION\TARGET CAPTURE

THE OBJECTIVE OF THIS PROJECT IS TO DEVELOP RAPID, HIGH YIELD METHODS FOR THE ISOLATION AND ENRICHMENT OF SPECIFIC NUCLEIC ACIDS FROM WHOLE BLOOD LYSATES. THESE METHODS WILL PROVIDE NUCLEIC ACID FREE OF BLOOD COMPONENTS WHICH ARE INHIBITORY TO THE POLYMERASE CHAIN REACTION (PCR). WHOLE BLOOD WILL BE LYSED INSTANTANEOUSLY BY CHEMICAL MEANS, AND THE NUCLEIC ACID WILL BE CAPTURED BY COMPLEMENTARY OLIGONUCLEOTIDES ATTACHED TO MAGNETIC BEADS. THE CAPTURED TARGETS WILL BE WASHED TO REMOVE EXTRANEOUS NUCLEIC ACIDS AND AGENTS THAT ARE INHIBITORY TO THE PCR. THE CAPTURED TARGETS WILL THEN BE DETECTED BY EITHER PCR AMPLIFICATION OF THE TARGETS DIRECTLY COUPLED TO THE BEADS, OR PCR AMPLIFICATION AFTER REMOVAL OF THE CAPTURED NUCLEIC ACIDS FROM THE BEADS. THE SENSITIVITY OF DETECTION OF TARGET MOLECULES PREPARED IN THIS MANNER WILL BE COMPARED TO THAT OF TARGET NUCLEIC ACIDS PREPARED BY CONVENTIONAL FICOLL DENSITY GRADIENT CENTRIFUGATION. IF THE TARGET CAPTURE APPROACH IS SUCCESSFUL, IT WILL SIGNIFICANTLY SIMPLIFY SAMPLE PREPARATION FOR THE PCR AND ALLOW MULTIPLE SAMPLES TO BE PROCESSED SIMULTANEOUSLY. THE LONG TERM OBJECTIVE IS TO DEVELOP AN AUTOMATED SAMPLE PREPARATION METHOD WHICH IS COMPATIBLE WITH THE REQUIREMENTS OF THE PCR.