chapter  2
9 Pages

Long PCR from Damaged DNA

I. Introduction 7

II. Use of Long PCR to Detect mtDNA Damage 8

A. DNA Isolation, Materials, and PCR Instrumentation 8

B. PCR Protocol 9

1. Principle of the Four-Primer Long PCR Protocol 9 2. PCR Conditions 9

3. Analysis of PCR Products and Interpretation of Data 10 C. Possible Pitfalls, Troubleshooting Tips, and Other Remarks 10

III. Use of Exonuclease III to Enhance Long PCR Amplification of Damaged DNA Templates 11

A. Effects of Exonuclease III on the Amplification of Damaged mtDNA Templates 11

B. Effects of Exonuclease III on the Amplification of Damaged Nuclear DNA Templates 12

1. Extraction of DNA, Materials, and PCR Instrumentation 12 2. PCR Conditions 12

3. Analysis of PCR Products and Interpretation of Data 13 C. Common Pitfalls, Troubleshooting Tips, and Specific Remarks 13

IV. Conclusions 14

References 15

I. INTRODUCTION

Several types of DNA lesions can hamper or block the progress of DNA polymerase during PCR amplification. Although the most common blocking lesions are DNA strand breaks and apurinic/apyrimidinic (AP) sites (also called abasic sites),1,2 other blocking lesions may exist.3 The larger the DNA sequence, the greater the probability that one or several blocking lesions will be present on each DNA molecule. Thus, whereas DNA must be extensively degraded to partially hamper amplification of a short DNA fragment, even mild DNA damage can totally prevent Long PCR amplification. Indeed, while DNA samples had to be heated at 99àC for more than 10 min to partly decrease amplification of a 316-bp mitochondrial DNA (mtDNA) sequence, heating DNA for only 2 or 3 min prevented the amplification of a 8600-bp mtDNA fragment.4,5

In the absence of genotoxic conditions or treatments, blocking DNA lesions are uncommon in living organisms. DNA strand breaks are absent or extremely rare, as these lesions are incompatible with cell life,6 and only 10 to 30 abasic sites are found per 106 nucleotides in normal rat or human tissues.7 These low physiological levels of blocking lesions are unlikely to significantly hamper Long PCR. However, the factors listed below can lead to more extensive DNA damage.