ABSTRACT
Polymerase chain reaction (PCR) is a widely practiced technique that is utilized in a number of applications in medical diagnostics, forensic DNA analysis, genome sequencing, and basic research. Using PCR, a dened nucleic acid sequence of interest is amplied using a thermostable DNA polymerase in combination with a pair of primers and 2′-deoxyribonucleoside 5′-triphosphates (dNTPs).1 The naturally occurring dNTPs are the most commonly used DNA polymerase substrates, where an equimolar mixture of the 5′-triphosphates of 2′-deoxyadenosine (dATP), 2′-deoxycytidine (dCTP), 2′-deoxyguanosine (dGTP), and 2′-deoxythymidine (dTTP) is routinely employed in PCR protocols. While the natural dNTPs are best suited for routine PCR, the inclusion of modied dNTPs into a PCR protocol can provide a wide range of benecial effects. As depicted in Figure 8.1a, there are three main components to a dNTP-the 2′-deoxyribose sugar, the nucleobase, and the triphosphate,
8.1 Introduction .......................................................................................................................... 103 8.1.1 Modied dNTPs to Improve the Specicity of PCR ................................................ 104 8.1.2 Modied dNTPs for Introduction of Mutations during PCR ................................... 107 8.1.3 Novel Base Pairs which Can Be Replicated during PCR ......................................... 109 8.1.4 Modied dNTPs for Introduction of Functionalities during PCR ........................... 112
8.2 Protocols ............................................................................................................................... 113 8.2.1 Basic PCR Protocol .................................................................................................. 113
8.2.1.1 Equipment .................................................................................................. 113 8.2.1.2 Supplies ...................................................................................................... 115
8.2.2 PCR Protocol I for Improved Specicity Using Hot Start dNTPs ........................... 115 8.2.2.1 Additional Supplies .................................................................................... 115
8.2.3 PCR Protocol II for Improved Specicity Using 7-deaza-dGTP for Amplication of GC-Rich Targets ...................................................................... 116 8.2.3.1 Additional Supplies .................................................................................... 116
8.2.4 Mutagenic PCR Protocol .......................................................................................... 117 8.2.4.1 Additional Supplies .................................................................................... 117
8.2.5 PCR Protocol with Novel Base Pairs ........................................................................ 117 8.2.5.1 Additional Supplies .................................................................................... 118
8.2.6 Protocol for Introduction of Functional Groups during PCR ................................... 118 8.2.6.1 Additional Supplies .................................................................................... 118
8.3 Concluding Remarks ............................................................................................................ 119 Acknowledgments .......................................................................................................................... 120 References ...................................................................................................................................... 120
each of which can be modied. Although there are a multitude of possibilities for chemical modi- cation within the dNTP framework, for practical use in PCR, it is critical that the dNTP modications do not signicantly reduce the efciency of nucleotide incorporation and recognition by DNA polymerase.
