Electrochemical primer extension for the detection of single nucleotide polymorphisms in the cardiomyopathy associated MYH7 gene

Article abstract of DOI:10.1039/c5cc07762a

We report the labelling of dideoxy nucleotides (ddNTPs) for use in electrochemical array based primer extension for the detection of single nucleotide polymorphisms (SNPs). The results confirm the extension of the immobilised primers for each of the four ddNTPs, representing a significant advance in achieving a cost-effective platform for screening of disease-specific SNPs.

Full text of DOI:10.1039/c5cc07762a

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Electrochemical primer extension for detection of single
nucleotide polymorphisms in the cardiomyopathy
associated MYH7 gene
Received 00th January 20xx
Accepted 00th January 20xx
a,c
c
c
a*
a,b*
A. M. Debela, S. Thorimbert , B. Hasenknopf , M. Ortiz and C. K. O´Sullivan,
DOI: 10.1039/x0xx00000x
We report the labelling of dideoxy nucleotides (ddNTPs) specific populations (e.g. to identify disease-associated
for use in electrochemical array based primer extension SNPs), which is predicted to result in patient stratification, for
for the detection of single nucleotide polymorphisms a more personalised approach to medicine.
(
SNPs). The results confirm extension of the immobilised
primers for each of the four ddNTPs, representing a Arrayed primer extension (APEX) is
significant advance in achieving a cost-effective platform genotyping method that exploits dideoxy nucleotides for
a
high throughput
for screening for disease-specific SNPs.
scanning known mutations over large regions of a DNA
sequence. Typically, the APEX procedure involves locus
5
The completion of the human genome project (HGP) has specific PCR amplification, followed by fragmentation using
paved the way for mapping diversity of the genome, thus uracil N-glycosylase. The fragmented PCR products are
helping to understand the genetic causes of inherited diseases denatured and hybridised to complementary capture probes
and susceptibility to drugs or environmental toxins. Over the that are surface-tethered on a glass array. Once hybridised,
past decade the development of new strategies for genotyping they serve as primers for template-dependent DNA
has attracted increasing interest, driven by the need for cost polymerase extension reactions using four fluorescently
effective and efficient strategies to take advantage of the labelled dideoxynucleotides. Imaging is followed by data
knowledge acquired during the HGP in order to assess a analysis to convert the fluorescence information into sequence
6
broad range of biological phenomena (e.g., genetic variation, data. The developed fluorescent APEX platform can
RNA expression, protein-DNA interactions and chromosome simultaneously interrogate many SNPs in a single multiplexed
7
conformation). Finally, the advance of technology across assay. Motivated by the high accuracy and specificity of the
diverse fields, including nucleotide biochemistry, polymerase fluorescent APEX, we have demonstrated electrochemical
engineering and computation, has facilitated the realisation of solid phase single base extension for SNP detection in the
1
alternative strategies
In the genome sequence there are cardiomyopathy associated in MYH7 gene. Electrochemical
individual variations that include single nucleotide primer extension has several advantages including cost-
polymorphisms (SNPs), insertions and deletions (indels), effective, simple-to-easy and portable instrumentation that
microsatellites (MSs), and differences in the methylation does not suffer from background light, as well as relatively
status of important regions (e.g. CpG islands). However, the inexpensive electrode arrays and well-established surface
majority of the variations are attributable to SNPs. SNPs are chemistries for automated probe immobilisation via spotting.
single base pair mutations in a genome that occur in at least The approach detailed here considerably simplifies previous
2
1
% of the total population. SNPs are attributable for 90% of reports of electrochemical detection of single base extension,
3
the genetic variations and the rest is attributable to insertions where SBE and detection are carried out separately, or use
or deletions of one or more 4bases, repeat length dUTPs which are problematic for homopolymers. Metallic
polymorphisms and rearrangements. The decoding of the nanoparticles (NPs) have also been used, where probes
8
human genome has revealed the presence of around 10 hybridise DNA containing the SNP to be interrogated, and
3
million SNPs (roughly
1
every 300-1000 bases). SNP following hybridisation mismatched bases are hybridised to
genotyping is of fundamental importance and vast metallic NPs specific for each base, which is then detected
9,10
international effort is currently being made using next- using stripping voltammetry.
generation sequencing to identify the location of SNPs in
In the work reported here, ddNTPs used were covalently
linked with four different redox active compounds with
distinguishable electrochemical signals (anthraquinone,
phenothiazine, methylene blue and ferrocene), at positions
favourable for enzymatic incorporation.
a.
Departament d’Enginyeria Química, Universitat Rovira i Virgili, Avinguda
b.Països Catalans, 26, 43007 Tarragona, Spain,
ICREA, Passeig Lluis Companys 23, 08010 Barcelona, Spain
c.
Sorbonne Universités, UPMC Univ Paris 06 Institut Parisien de Chimie
Moléculaire, UMR CNRS 8232, 4 place Jussieu, 75005 Paris, France.
*
Corresponding author e-mails: mayreli.ortiz@urv.cat; ckosulli@etse.urv.es
Electronic Supplementary Information (ESI) available: [Details of sequences
and experimental procedures used]. See DOI: 10.1039/x0xx00000x
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Fig. 1 The redox active dideoxy nucleotide triphosphates used for
electrochemical primer extension assays. (AQ: anthraquinone (-0.40
V), MB: methylene blue (-0.20 V), Fc: ferrocene (0.50 V) and PTZ:
phenothiazine (0.60 V)) All potentials vs Ag/AgCl.
The four different redox labels were chosen based on their
distinctive redox potentials, allowing clear discrimination
between the labels (Fig. 1). Carboxyl functionalised ferrocene
(
Fc), anthraquinone (AQ) and methylene blue (MB) were
purchased from commercial sources, while phenothiazine was
functionalised with an alkyl arm bearing a carboxyl terminus
following the stepwise reaction protocol described in Figure
SI1. These carboxyl derivatives were used for functionalising
propargyl amine bearing ddNTPs via amide bond formation, to
achieve compounds for electrochemical solid phase primer
extension (éPEX) reaction. The labelled ddNTPs were
characterised using NMR and ATR FT-IR (Supporting Fig.
2
Top: Surface-tethered DNA probes hybridise to target
Information). As can be seen in Fig. 2, éPEX consists of sequences one base prior to the SNP-site to be interrogated, followed
hybridisation of the template to a surface-tethered probe, by incorporation of redox-labelled ddNTP. Bottom: Dependent on the
specific base present at the SNP site, different ddNTPs will be
followed by enzymatic incorporation of a label modified
incorporated, and the incorporated ddNTP can be identified via the
ddNTP, which terminates elongation, thus only extending the
DPV signal of the redox label. [Note: The schematic above shows the
possibility for each potential base present at the SNP site, but only
one of the four labelled ddNTPs will be incorporated at each SNP
primer by one single base at the known SNP location.
Subsequent to primer extension, electrochemical interrogation
of the incorporated labels was performed by monitoring the
redox reaction using differential pulse voltammetry (DPV) in
potential windows corresponding to each of the labels.
site.]
2
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behaviour. The results obtained thus confirm VitehweArtiscluerOfanlcinee
confinement of the incorporated labelled ddNTPs. Following
DOI: 10.1039/C5CC07762A
the representation of Fig. 2, exploiting the electroactive
properties of the modified ddNTPs, differential pulse
voltammetry (DPV) was used to study the accuracy and
specificity of their incorporation. As shown in Fig. 4, when the
éPEX reaction was performed in the presence of the four
labelled ddNTPs, the DPV signal corresponding specifically to
the incorporated ddNTP was significantly higher than the
signals from the non-specific labelled ddNTPs, thus confirming
the fidelity of the polymerase incorporating the correct
modified ddNTP. Due to electrostatic / groove interactions with
DNA, anthraquinone, methylene blue and phenothiazine show
slightly higher non-specific signals as compared to ferrocene.
Methylene blue is known to interact with free guanines in
16
DNA.
However, the signal attributed to the incorporated
base is significantly higher than the non-specific background.
Fig. 3 Left: Cyclic voltammograms (in 10 mM Tris buffer + 0.5 M NaCl,
pH 7) for surface tethered DNA duplexes incorporated with the four
different redox-labelled ddNTP as follows: a) AQ-ddATP, b) MB-
ddUTP, c) Fc-ddGTP and d) PTZ-ddCTP. Right: The corresponding
plots of the cathodic peak current, (Ipc) vs different scan rate, (υ).
Modified polymerases have been shown to be highly tolerant
to nucleotide modifications with various groups at position 5 of
pyrimidine bases (C and U) and position 7 of purine bases (A
and G). Various reports have detailed the enzymatic
Fig. 4 DPV signals recorded after four éPEX reactions performed
each in the presence of the four labelled ddNTPs. The main signal in
each case corresponds to the specific incorporation of (a) AQ-ddATP,
(
b) MB-ddUTP, (c) Fc-ddGTP and (d) PTZ-ddCTP
8,11,12
incorporation of dNTPs labelled with ferrocene,
8b,13,14
14
anthraquinone,
phenothiazine, and gel electrophoresis
We have demonstrated the design, synthesis and
characterisation of four electroactive labelled ddNTPs with
distinctive redox potentials. The synthesised labelled ddNTPs
were subsequently used for electrochemical solid-phase
primer extension assays. These éPEX assays can be adopted
for the detection of multiple mutations/SNPs using arrays of
electrodes, offering an excellent platform for the detection of
disease-specific SNPs, addressing the future medical
paradigm of patient stratification for personalised medicine.
Future work will focus on the use of alternate redox labels to
reduce the background signal and expanding to simultaneous
interrogation of multiple SNP sites.
has been used to confirm the incorporation of the modified
dNTPs. In the work reported here, where we exploit surface
tethered probes and primer extension reactions, the
incorporation of the modified ddNTP can be demonstrated
using cyclic voltammetry. Target DNA sequences varying in
length from 24-48 bases were used, but the length of the
single stranded target DNA to be interrogated is not expected
to have any effect on the electrochemical sensitivity as the
labelled ddNTP will be incorporated on to the surface-tethered
probe following hybridisation, the duplex inferring inflexibility
and always positioning the incorporated redox label at the
same distance from the electrode surface.
Acknowledgement:
Confirmation of the surface confinement of the incorporated
redox labelled ddNTPs was demonstrated by carrying out
cyclic voltammograms at different scan rates (Fig. 3). The
Cardioscreening, Plan Nacional Subdirección General
Proyectos de Investigación, BIO2011-25369. This project has
also received funding from the European Union's Horizon
15
peak current is proportional to the scan rate (υ) (the solid
lines in the graph of right column are linear fits based on the 2020 research and innovation programme under the Marie
1
/2
method of least squares) in contrast to υ dependence, which Sklodowska-Curie grant agreement No 659211"
arises from diffusing species following the Nernstian
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