Biomedical Engineering Reference
In-Depth Information
time quantitative polymerase chain reaction (PCR), fetal DNA can be reliably
detected in maternal plasma between the fifth to seventh week of gestation
(
2-4
)
.
Circulating fetal DNA concentration increases with progression of pregnancy
and disappears from maternal plasma rapidly after delivery, with a median half-
life of 16.3 min
(
2
,
5-7
)
. Hence, circulating fetal DNA represents a promising
source of fetal genetic material for prenatal diagnosis while being accessible
simply by maternal venesection.
Soon after its discovery, numerous potential applications have been reported
for the analysis of fetal DNA in maternal plasma. Abnormal concentrations of
circulating fetal DNA had been reported for preeclampsia
(
8
,
9
)
, fetal aneup-
loidy
(
10
,
11
)
, preterm labor
(
12
)
, fetal hemorrhage
(
13
)
, invasive placentation
(
14
,
15
)
, hyperemesis gravidarum
(
16
)
, and oligohydramnios
(
17
)
. Mutation
detection through circulating fetal DNA analysis had been reported for a num-
ber of hereditary traits including fetal rhesus D status
(
18
,
19
)
,
-thalassemia
(
20
,
21
)
, congenital adrenal hyperplasia
(
22
)
, and achondroplasia
(
23
)
.
Because of the relative high abundance, fetal DNA can be readily detected
in maternal plasma with PCR amplification, particularly when a number of
preanalytical and analytical considerations are taken into account
(
24
)
. These
considerations include processes involving the choice of specimen, timing and
procedures for blood processing, design and format of assay system, and anti-
contamination procedures.
β
2. Materials
2.1. Sample Collection
Ethylenediamine tetraacetic acid (EDTA)-containing blood collection tubes
for plasma collection.
2.2. DNA Extraction
1. Absolute ethanol.
2. QIAamp Blood Mini Kit (Qiagen, Hilden, Germany).
a. Protease.
b. Lysis buffer.
c. Spin columns.
d. Collection tubes.
e. Wash buffer 1.
f. Wash buffer 2.
3. Deionized water (dH
2
O).
2.3. Real-Time Quantitative PCR
2.3.1. Amplification Reagents
(2)
1.
Primers (
see
Note 1
):
