Biomedical Engineering Reference
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fragments (Chan et al. 2004 ). Efforts to increase the relative proportion of fetal
DNA compared to the larger maternal fraction have also included the use of form-
aldehyde, as a fi xative, to prevent lysis of maternal cells during the isolation of the
maternal plasma (Dhallan et al. 2004 ). The formaldehyde enrichment technique,
however, has not been reproducible by other laboratories.
Different methodologies applied for the detection of cffDNA include conven-
tional PCR, restriction analysis, quantitative fl uorescence real-time PCR (QF-PCR),
and automated sequencing (Bustamante-Aragones et al. 2008 ; Chiu et al. 2002a , b ;
Ding et al. 2004 ; Li et al. 2007 ; Saito et al. 2000 ). Since QF-PCR is more sensitive
compared to conventional PCR, enabling the detection of very low copy numbers of
DNA, it represents the optimal method for reliable NIPD. The main advantage of
QF-PCR is that it is quantitative and collects data in the exponential growth phase
of the reaction, which is the most specifi c and precise one. The technique is less
time-consuming and offers an extra level of protection against contamination.
A wide range of Ct values in each QF-PCR and poor repeatability of some replicates
are reported, partly due to the variability of target copy number in maternal plasma.
It is, therefore, recommended to perform several replicates from each maternal sam-
ple in order to increase the probability of fetal DNA detection and to avoid false
negative results (Minon et al. 2008 ). Recently, new sophisticated molecular tech-
niques, such as NGS, have emerged and have been applied to the fi eld of NIPD (Fan
et al. 2008 ). They have higher sensitivity, but the expensive and complicated han-
dling processes are required by these techniques. NGS can analyze the nucleotide
sequences of millions of DNA molecules in each run. The capacity of NGS to dif-
ferentiate small quantitative alterations in genomic distributions of chromosomes
has allowed detection of higher number of chromosome 21 sequences in trisomy 21
pregnancies as compared to euploid pregnancies.
Principle of Aneuploidy Detection with
The principle of the use of NGS for noninvasive fetal chromosomal aneuploidy
detection in maternal plasma is shown in Fig. 5.1 (Chiu et al. 2008 ). Maternal
plasma DNA (maternal and fetal) is naturally fragmented and further fragmentation
is unnecessary for the next steps (Chan et al. 2004 ). The plasma DNA fragments are
sequenced by an Illumina sequencer and processed by the Effi cient Large-Scale
Alignment of Nucleotide Databases (ELAND) software to determine the chromo-
somal origin, but the details about their gene-specifi c location are not required.
Quantifi cation of the number of sequence reads originating from any particular
chromosome is performed for each human chromosome. Chiu et al. counted only
sequences that could be mapped to just one location, unique sequences denoted as
U0-1-0-0 on the basis of values in a number of fi elds in the data output fi les of the
ELAND sequence alignment software, in the repeat-masked reference human
genome with no mismatch. The percentage contribution of unique sequences
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