POLYMERASE CHAIN REACTION: Applications in Environmental Microbiology

INTRODUCTION ........ .. .. . . . ........ . 138 THE POLYMERASE CHAIN REACTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 1 38 PCR Reaction Mixture . . . . . . . .. . ........ 138 PCR Reaction Conditions ........ . . . . . . . . ....... . . . . 140 Multiplex PCR . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .. . . . . . . . .. ... ... .. ....... . . . . . 1 4 1 Enhancing PCR Efficiency .. . ... . . . . . . . . . . . . 141 ISOLATION OF ENVIRONMENTAL NUCLEIC ACIDS FOR PCR . . . . . .. . . . . ....... . . . 142 Isolation of Nucleic Acids from Aquatic Environments.. . .. . . .... ........ . . ..... 142 Isolation of Nucleic Acids from Soil and Sediment . . .. . . . ....... . . 143 DETECTION OF PCR PRODUCTS .... . . . . . . ..... . . . . . . .... . . . . .. . . . . . . . . . . . . . . . . . . . . . ..... . . . . . 144 Capture Probes-Reverse Hybridization . .... . . . . . .. . .... . . . . . . . . 146 USE OF PCR TO DETECT MICROORGANISMS IN ENVIRONMENTAL SAMPLES 147 Detection of Genetically t;ngineered Microorganisms .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Detection of Indicator Organisms and Pathogens... . . . . . . . . . ...... . . . . . . . . . . . . ....... . . . . . 148 QUANTITATION OF PCR-AMPLIFIED PRODUCT . . . . . . . . . . . . . . . . . .. ...... . . . . .... . . . ..... 149 Quantification of DNA Target Sequence . . . . .. . ... . .. . . . . . . .. . . . ....... .. . . .. . . .. . ...... . . . . . 149 Quantification of mRNA .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 50 USE OF PCR TO ISOLATE AND CLONE SPECIFIC DNA SEQUENCES ... . . ....... 1 52 Use of PCR to Analyze Ribosomal RNA Sequences ....... . . . . . . . . . ........ . . . 152 Cloning DNA and Creating Gene Probes Using Degenerate Primers ... ........ . . . ... 153 CONCLUSION . . . . . . . . . . .. . . . . . .... . ......... 1 55