Biomedical Engineering Reference
In-Depth Information
a.
SRY
:
SRY-109F: 5'-TGG CGA TTA AGT CAA ATT CGC-3'.
SRY-245R: 5'-CCC CCT AGT ACC CTG ACA ATG TAT T-3'.
b.
β
-globin
:
β
-globin-354F: 5'-GTG CAC CTG ACT CCT GAG GAG A-3'.
β
-globin-455R: 5'-CCT TGA TAC CAA CCT GCC CAG-3'.
2.
Dual-labeled fluorescent probes (
see
Note 1
):
a.
SRY
:
SRY-142T: 5'-(FAM)AGC AGT AGA GCA GTC AGG GAG GCA
GA(TAMRA)-3'.
b.
β
-globin
:
-globin-402T: 5'-(FAM)AAG GTG AAC GTG GAT GAA GTT GGT
GG(TAMRA)-3'.
where FAM is 6-carboxy-fluorescein; TAMRA is 6-carboxy-tetramethyl-
rhodamine.
β
3.
Calibrator: male genomic DNA.
4.
TaqMan PCR core reagent kit (Applied Biosystems, Foster City, CA).
a. 10X buffer A.
b. MgCl
2
.
c. dATP, dCTP, dGTP, dUTP.
d. Ampli
Taq
Gold.
e. AmpErase uracil
N
-glycosylase.
5.
dH
2
O.
2.3.2. Instrumentation for Quantitative Analysis
ABI Prism 7700 Sequence Detector (Applied Biosystems).
3. Methods
The methods described below outline the procedures for (1) sample collec-
tion, (2) sample processing, (3) maternal plasma DNA extraction, (4) construc-
tion of calibration curves, (5) real-time quantitative PCR analysis, (6) data
interpretation, and (7) prevention of contamination.
3.1. Sample Collection
Collect at least 2 mL of maternal venous blood into EDTA tubes (
see
Note 2
).
3.2. Sample Processing
1.
Maternal blood samples may be kept at room temperature or 4°C until processing
(
see
Note 3
).
2.
Process maternal whole blood within 6 h to obtain plasma (
see
Note 3
).
3.
Centrifuge maternal whole blood at 1600
g
for 10 min.
4.
Transfer supernatant to clean polypropylene tubes with due caution not to disturb
the underlying buffy coat layer.
