Biomedical Engineering Reference
In-Depth Information
4.
Other DNA/RNA quantifications requiring absolute measurements and/or single
base specificity.
2. Materials
1.
cDNA or DNA samples.
2.
Water (DNase and RNase free, 0.1
µ
m filtered, Sigma, W4502).
3.
Hot Start
Taq
Polymerase (Qiagen).
4.
Hot Start
Taq
Buffer (Qiagen).
5.
MgCl
2
.
6
SAP (Sequenom).
7.
ThermoSequenase (Sequenom).
8.
dNTP mixture (GIBCO), for PCR, 25 m
M
each.
9.
ddNTP/dNTP mixtures (Sequenom). Typically, three different ddNTPs and one
dNTP are mixed together at equal molar concentrations.
10.
PCR and extension primers (IDT Technologies).
11.
Oligonucleotide standard (60-80 bases), polyacrylamide gel electrophoresis
(PAGE)-purified, concentration determined by absorbance at 260 nm, stored at
10
µ
M
in -80°C.
12.
hME buffer (Sequenom).
13.
384- or 96-well microplate (Marsh Biomedical Products) and plate seal (Applied
Biosystems, cat. no. 4306311).
14.
96-channel Multimek auto-pipet (Beckman Coulter) for high-throughput analy-
sis or a 12-channel electronic pipet (0.5-10
µ
L, cat. no. E12-10, Rainin) for low-
and medium-throughput analysis.
15.
96-Well SpectroCLEAN plate, SpectroCLEAN resin and scraper (Sequenom).
16.
384-format SpectroCHIP prespotted with 3-hydroxypicolinic acid (Sequenom).
17.
SpectroPOINT nanodispenser (Sequenom).
18.
A modified Biflex (or Autoflex) MALDI-TOF mass spectrometer with Quantita-
tive Gene Analysis (formerly called Allelotyping) software (Sequenom).
3. Methods
The methods described here include (1) rcPCR assay design and (2) rcPCR
protocol including competitive PCR, SAP treatment, primer extension reac-
tion, post-PCR sample processing and nanodispensing, and MALDI-TOF MS.
Using the commercially available MassARRAY (Sequenom) system, which is
bundled with all necessary softwares for the most efficient and accurate analy-
ses, is highly recommended. Expert users may also be able to replicate the
applications in any MALDI-TOF MS-based platforms.
3.1. rcPCR Assay Design
3.1.1. rcPCR for DNA Quantification
This section provides a generic design for quantifying any genomic DNA
sequence (i.e., to quantify the presence of an X-chromosome sequence). The
