Download Supplemental Data Methods

SUPPLEMENTAL DATA METHODS
1. Identification by WAVE analysis of heteroduplex DNA containing
rs2187247
a. Pretreatment. PCR fragments (262 bp of C21orf105 cDNA generated by RTPCR) were treated using exonuclease I and shrimp alkaline phosphatase
(ExoSAP-IT) (USB) followed by Qiagen column purification to remove
deoxynucleotides and primers. Both procedures were done according to the
manufacturers instructions, except that incubation with ExoSAP-IT was done
for 60 min. RT-PCR was done as described elsewhere (1). Purified DNA,
eluted in MilliQ water, was heated at 95oC for 5 min and cooled to 25oC at a
rate of 1.5oC/min to allow formation of heteroduplexes.
b. WAVE procedure. PCR product (10 l) (100-500 ng DNA) was then loaded
for partially denaturing HPLC analysis using the WAVE 3500 HT system
(Transgenomic). Elution was done with a gradient mixture of buffer A (0.1
mol/L triethylammonium acetate) and buffer B (0.1 mol/L triethylammonium
acetate, 250 ml/L acetonitrile) (Transgenomic). Running temperature and
percentage of buffer B were 62.7oC and 55.5 %, respectively. The temperature
optimal for partially denaturing HPLC conditions was calculated using the
NAVIGATOR Software. Initial tests included runs at 0.5oC above and below
this temperature. Detailled running conditions are shown below.
c. Data analysis. DNA fragment elution profiles were captured online using
UV detection at 260 nm and visually displayed using the Transgenomic
1
WAVEMAKER software. Chromatograms were analyzed for the presence of
rs2187247 and compared with the results obtained by DNA sequencing.
TABLE
Running conditions
Time
%A
%B
Loading
0
47.5
52.5
Start gradient
0.1
44.5
55.5
Stop gradient
2.1
34.5
65.5
Start clean
2.2
47.5
52.5
Stop clean
2.3
47.5
52.5
Start equilibration 2.4
47.5
52.5
Stop equilibration 2.5
47.5
52.5
2. Quantification of allelic expression ratios of C21orf105 by quencher
extension
a. Pretreatment. PCR fragments (262 bp of C21orf105 cDNA generated by RTPCR) were treated using exonuclease I and shrimp alkaline phosphatase
(ExoSAP-IT) (USB) followed by Qiagen column purification to remove
deoxynucleotides and primers. Both procedures were done according to the
2
manufacturers instructions, except that incubation with ExoSAP-IT was done
for 60 min. RT-PCR was done as described elsewhere (1).
b. Standard curves. To generate standard curves, PCR fragments containing
either the A or C allele of rs2187247 were cloned into pTOPO vectors. Plasmids
containing either the A or C allele, as confirmed by sequencing, were purified
by Qiagen column affinity purification and subjected to PCR. Amplicons were
treated (ExoSAP-IT) and purified as above (Qiagen), quantified using
A260/280 ratios (Nanodrop) and used to generate standard curves. Standard
curves included 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 mixtures of
the respective alleles of rs2187247 with concentrations ranging between 10 and
100 ng DNA.
c. Quencher extension reaction. The treated, purified products were used as
templates in the Quencher Extension reaction (QEXT) using a modification of
the procedure of Rudi and coworkers (2, 3). For this, 5 l PCR product (50 ng)
was added to 1 x Thermosequenase reaction buffer, 1 pmol of 5’-6-FAM
labeled probe (5’-AGC TTT TTG TGA GGG TCA G-3’) (Eurogentec), TAMRAddUTP (4 pmol/L) or TAMRA-ddGTP (0.4 pmol/l) (Perkin Elmer), ROX
reference dye (1 l) (Applied Biosystems) and 8 U Thermosequenase
(Amersham Biosciences) in a total volume of 25 l. The probe is
complementary to the coding strand and directly flanks the single nucleotide
polymorphism (rs2187247). Probe design (length, strand) were optimized
using Oligo software with selection for absent or low frequencies of duplex-,
hairpin- and 5’-overhang formation. Following incubation for 3 min at 95oC,
3
sequence-specific extension was done by thermocycling for 40 cycles using the
ABI 7300 Real-Time PCR system. Each cycle consisted of denaturation at 95oC
for 30 sec and labeling at 55oC for 1 min.
Positive controls consisted of 50 ng of AA or AC samples for QEXT reactions
performed for the A allele using TAMRA-ddUTP. Positive controls consisted
of 50 ng of CC or AC samples for QEXT reactions performed for the C allele
using TAMRA-ddGTP. Negative controls consisted of homozygous DNA (50
ng) containing either the A or C allele for QEXT reactions performed with
TAMRA-ddGTP or – ddUTP, respectively.
d. Data analysis. The signals were measured in relative fluorescence units
(RFU) and expressed as Rn (normalized reporter signal) as measured for each
cycle during the denaturation phase. The Rn value is the fluorescence emission
intensity of the reporter dye (6-FAM at 541 nm) divided by the fluorescence
emission intensity of the passive reference dye (ROX at 585 nm). For the
combination used (FAM reporter, TAMRA quencher), the increase in
extension, i.e. amount of SNP present, correlates with a decrease in
fluorescence of the reporter (FAM) molecule. Regression curves were obtained
using polynomial regression employing the normalized reporter (FAM)
signals. The initial slopes (15 cycles) of the regression curves were used for
quantification. For this, the raw data (component data), are exported to Excel.
The FAM signal (probe) divided by the ROX signal (internal standard)
generates Rn. The maximum (Rnmax) is calculated for all Rn values.
Subtraction of the Rn value (first cycles) from the maximum generates the
4
correction factor for each sample (Cfa). This is applied to the Rn value for each
cycle. All these values are exported to Prism and used for calculation.
Calculation of slope ratios
For each sample the slope ratio (ddUTP ‘curve’: ddGTP ‘curve’ or A : C) of the
regression curves were calculated. From the results of the duplicates the mean
was calculated. This number was compared with the result of diploid
calibration standard sample (AC) from the same run. The result of the AC was
used as the median, by definition 1. The results of the ACC and AAC and the
placenta samples were expressed as multiples of the median (MoM). All
heterozygous triploid calibration standard samples and trisomy 21 placenta
samples showed deviated allelic ratios from that of the diploid calibration
standard sample and euploid placenta. We defined the interval between 0.7
and 1.3 MoM as the ‘normal’ or diploid range.
3. Application of WAVE and QEXT on first trimester plasma samples
Placental RNA was isolated and subjected to RT-PCR as described previously
(1) with modifications (4). Prior to WAVE and QEXT analysis, the total amount
of the ExoSap-treated, purified amplicons was calculated using the Agilent
2100 BioAnalyzer. For first trimester plasma samples (weeks 9-14), the average
total amount of specific product available at the start of the WAVE and QEXt
procedures is about 5-10 fold higher than the minimal amount needed.
5
Supplemental references
1.
Oudejans CB, Go AT, Visser A, Mulders MA, Westerman BA,
Blankenstein MA, van Vugt JM. Detection of chromosome 21-encoded
mRNA of placental origin in maternal plasma. Clin Chem 2003;49:14459.
2.
Rudi K, Holck AL. Real-time closed tube single nucleotide
polymorphism (SNP) quantification in pooled samples by quencher
extension (QEXT). Nucleic Acids Res. 2003;31:e117.
3.
Rudi K, Zimonja M, Hannevik SE, Dromtorp SM. Multiplex real-time
single nucleotide polymorphism detection and quantification by
quencher extension. Biotechniques 2006;40:323-9.
4.
Go ATJI, Visser A, Mulders MAM, Twisk JWR, Blankenstein MA, van
Vugt JMG, Oudejans CBM. C21orf105, a chromosome 21-encoded
mRNA, is not a discriminative marker gene for prediction of Down
syndrome in maternal plasma. Pren. Diagn. 2007;27: 146-9.
6