Biomedical Engineering Reference
In-Depth Information
6
Qualitative and Quantitative DNA and RNA Analysis
by Matrix-Assisted Laser Desorption/Ionization
Time-of-Flight Mass Spectrometry
Chunming Ding
Summary
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS) gives extremely precise reading of mass-to-charge ratios (two
analytes differ by 1 Da can be distinguished) and provides high sensitivity (less than 1
fmole of a DNA oligonucleotide can be detected), allowing DNA quantifications with
single base specificity and single DNA molecule sensitivity (coupled with polymerase
chain reaction [PCR]). To quantify a DNA sequence of interest, a competitive synthetic
(60-80 bases) oligonucleotide standard with an artificial single base mutation in the
middle is introduced, and these two virtually identical sequences are co-amplified by
PCR. A third primer (extension primer) is designed to anneal to the region immediately
upstream of the mutation site. Depending on the specific mutation introduced and the
ddNTP/dNTP mixtures used, either one or two bases are added to the extension primer to
produce two extension products from the two templates. Last, the two extension products
are detected and quantified by high-throughput MALDI-TOF MS. In addition, with an
improved primer extension method called single allele base extension reaction (SABER),
rare mutant DNA can be robustly detected even when normal DNA is present at 50-fold
or more than the DNA mutants.
Key Words:
DNA mutation; single nucleotide polymorphism (SNP); gene expres-
sion; DNA quantification; matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS); competitive PCR; primer extension; real-competitive
PCR (rcPCR); single allele base extension reaction (SABER); prenatal diagnosis.
1. Introduction
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS), originally developed for peptide analysis
(
1
)
, is extremely
precise in determining the mass-to-charge ratios (single Dalton resolution
[
2
]
)
and very sensitive (0.7 fmole DNA oligonucleotide
[
3
]
), yet remains poorly
