Analysis of Diverse Plant Species using Mass Spectrometric Cleaved Amplified Polymorphic Sequences (MS-CAPS) and Improvement of PCR Efficiency by Addition of Cysteine

摘要

The applicability of mass spectrometric cleaved amplified polymorphic sequences (MS-CAPS) was evaluated in several plant species. This method consists of genomic DNA extraction from plant tissues, polymerase chain reaction (PCR) amplification of a specific genetic region, enzymatic digestion of amplicons, and followed by rapid analysis of single nucleotide polymorphisms (SNPs) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Crude extracts obtained by homogenizing plant tissues in water were used as templates for short PCR amplifications for MS-CAPS analysis. For most plant species tested, these crude extracts could be used directly as templates for PCR. However, extracts from lettuce leaves and stems showed enzymatic browning as a result of polyphenol oxidase (PPO) activity, and were not suitable PCR templates. The addition of cysteine to the homogenizing solution inhibited enzymatic browning and did not affect the other MS-CAPS procedures, including PCR amplification, uracil-DNA glycosylase treatments, or MALDI-TOF MS analysis. Thus, this method inhibits PPO in crude extracts, allowing them to be used directly for MS-CAPS analysis.