Biomedical Engineering Reference
In-Depth Information
tions or loss, such as are frequently found in cancer. It is worth noting that
although the quantitative fluorescent PCR analysis of STRs has been estab-
lished and clinically implemented for the rapid prenatal analysis of the most
common aneuploidies, the use of real-time PCR does have a few advantages in
that it is more amenable to automation and, because it is a closed system, is less
prone to contamination.
2. Materials
2.1. DNA Extraction
1.
For the extraction of DNA from the amniotic fluid samples, Chelex 100 resin
(biotechnology grade 100-200 mesh sodium form) from Bio-Rad (Reinach, Swit-
zerland, cat. no. 142-1253) was used.
2.
It is recommended that a highly pure water source, such as Analar water (molecular
biology grade, VWR International), be used. Alternatively, Milli-Q water deion-
ized with the Elgastat Maxima (Kleiner AG, Wohlen, Switzerland) can be used.
2.2. Real-Time PCR
1.
For our real-time PCR analysis, we used an ABI PRISM 7000 Sequence Detec-
tion System (SDS7000) (
see
Note 1
).
2.
For the PCR reactions, we used MicroAmp Optical 96-well reaction plates
(Applied Biosystems, Switzerland, cat. no. 4306737) and the ABI PRISM Opti-
cal adhesive covers (cat. no. 4311971) (
see
Note 2
).
3.
The reactions were carried using the TaqMan Universal PCR master mix (cat. no.
4304437) containing Ampli
Taq
Gold DNA Polymerase, AmpErase uracil-
N
-gly-
cosylase (UNG), dNTPs with dUTP, and passive Reference 1 (ROX fluorescent
dye) (
see
Note 3
).
4.
Primer and probe sequences are listed in
Table 2
(
see
Note 4
).
5.
The best results were obtained using TaqMan minor groove-binding (MGB)
probes (cat. no. 43160324) from ABI. These are 3'-labeled with a minor groove
binder and a nonfluorescent quencher (
see
Note 5
). The chromosome 18 probe
is 5'-labeled with the fluorescent dye VIC, and the chromosome 21 probe is
5'-labeled with FAM. It is advisable to store these probes as 5-
M
aliquots at
-20°C, at which they are stable for over 1 yr. If used rapidly (within 1 mo), the
probes can be stored at 4°C. In this case, aliquots of both probes should be used
and stored in parallel. Avoid exposure to light.
µ
6.
In our experience, the best results were obtained using high-performance liquid
chromoatography (HPLC)-purified primers (Microsynth, Switzerland) (
see
Note 6
). These can be stored as 10-
µ
M
aliquots at -20°C. Primers stored at 7°C
are stable for several months.
