Biomedical Engineering Reference
In-Depth Information
quantitative. The poor quantitative ability of MALDI-TOF MS is mainly owing
to ionization/desorption differences between amino acid residues (or nucle-
otide residues
[
4
]
), and uneven matrix-analyte crystal formations. MALDI-
TOF MS is extremely fast (a few seconds for each sample) and has been highly
automated
(
3
,
5
,
6
)
. As a result, MALDI-TOF MS is most widely used in high-
throughput qualitative analyses, such as protein sequencing
(
7
)
and single
nucleotide polymorphism (SNP) scoring
(
3
,
8
)
.
Although it is hard to quantify protein or DNA on the basis of absolute mass
spectrometric signal intensity, it is highly accurate and reproducible to quan-
tify the ratios of two chemically identical (or very similar) proteins or oligo-
nucleotides in a single mass spectrum. Recently, MALDI-TOF MS has been
used for high-throughput protein
(
9
)
and DNA/RNA
(
10
,
11
)
quantifications.
This chapter provides a working protocol, which combines competitive poly-
merase chain reaction (PCR) and MALDI-TOF MS, for a DNA/RNA quantifi-
cation technique known as real competitive PCR (rcPCR). In addition, DNA
mutation analyses can be carried out simultaneously.
To quantify a DNA sequence of interest, a synthetic (60-80 bases)
oligonucleotide standard with an artificial single base mutation in the middle is
co-amplified with a virtually identical sequence of interest by PCR. Shrimp
alkaline phosphatase (SAP) is used to remove excess dNTPs. A third primer
(extension primer) is designed to anneal to a location right next to the mutation
site. Depending on the specific mutation introduced and the ddNTP/dNTP mix-
tures used, either one or two bases are added to the extension primer, produc-
ing two extension products from the two templates. The two extension products
are then detected and quantified by high-throughput MALDI-TOF MS (
Fig. 1
).
Because DNA quantification by rcPCR is based on its ability to distinguish
two DNA sequences differing by a single base, it is also possible to detect and
quantify mutants (in the presence of wild-type DNA sequences). A slightly
more complicated scheme is needed for assay design, because three competing
DNA sequences (standard DNA, wild type DNA, and mutant DNA) are present
in the system. However, the experimental procedure remains essentially the
same.
In summary, this technique can be used in high-throughput analyses such as:
1.
Gene expression analysis. In particular, one can study minor (20-50% down- or
upregulation in gene expression), yet biologically significant changes, and allele-
specific expression.
2.
DNA quantification for analyzing loss of heterozygosity (LOH), trisomies, mono-
somies, and gene amplification.
3.
Mutant DNA detection and quantification in the background of wild-type DNA
sequences.
