SCIENTIFIC REPORT
Peptide nucleic acids as probes to detect
disease-specific point mutations using
Biacore’s SPR technology
Roberto Gambari
Biotechnology Center, Ferrara University, Ferrara, Italy.
We report the use of peptide nucleic acids (PNAs) to efficiently identify
W1282X Cystic Fibrosis point mutations in PCR-generated targets using
Biacore’s SPR technology. The results demonstrate the application of using
PNAs in Biacore systems for the molecular diagnosis of human hereditary
mutations.
W
Figure 1
Experimental strategy: in panel A, biotinylated
target ODNs (either normal or carrying the CF
W1282X mutation) were immobilized onto
two different flow cells. In panel B, target DNA
was produced by PCR using a biotinylated
primer and DNA substrates from normal,
heterozygous and homozygous W1282X
samples. Biotinylated PCR products were
injected onto three different flow cells and
single stranded DNA was readily obtained
with 5 µl pulses with 50 mM NaOH. The
sequence of the N-W1282X probe was 5'TCCTCCACT-3' (DNA) and H-TCCTCCACTNH2 (PNA), the sequence of M-W1282X
probe was 5'-TCCTTCACT-3' (DNA) and HTCCTTCACT-NH2 (PNA) (6).
e have proposed real-time monitoring of DNA:DNA hybridization using Biacore’s SPR technology for the detection of HIV-1 infection (1),
genetically-modified organisms (2) and mutations of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, ∆F508 (3) and W1282X (4). Peptide nucleic
acids (PNAs) (5), in which the sugar-phosphate backbone is replaced by N-(2-aminoethyl)
glycine units, are of interest in such applications. Hybridization of PNAs to complementary DNA is efficient, generates Watson-Crick
double helices, and is largely independent of
limitations imposed by structural artifacts on
target DNA (5). PNAs are predicted to bind to
single stranded PCR products more efficiently
than oligodeoxyribonucleotides (ODN) (6),
and to better enable point mutations to be
detected. Using Biacore’s SPR technology, we
found that ODN probes containing 15-20
nucleotides hybridize efficiently but do not
discriminate between fully matched and mismatched target DNA (3, 4), while shorter
A
B
DNA
or PNA
probes
Target
oligonucleotides
(21-mer)
Target
PCR
product
EFFICIENT AND SPECIFIC BINDING OF PNA PROBES
Sensor chip
PAGE 14
DNA
or PNA
probes
ODNs, while discriminating between targets
containing point mutations, are less efficient
in hybridization. By contrast, even short PNAs
are predicted to efficiently hybridize to target
DNA, the melting temperature of DNA:PNA
hybrids being higher than that of DNA:DNA
hybrids (5) due to the fact that PNAs are not
negatively charged.
We have recently described the use of PNA
probes in Biacore® systems for the detection of
the tryptophan 1282 -> TER mutation of the
CFTR gene (6). The Biacore 1000 analytical
system, Sensor Chip SA, precoated with streptavidin, and the running buffer HEPES buffered
saline-EP (HBS-EP), all from Biacore AB,
Uppsala, Sweden, were used. The experiments
were conducted at 25°C at a flow rate of
5 µl/min.
Here we review the results obtained in two
different experimental strategies based on
Biacore’s SPR technology. In the first strategy,
biotinylated target DNAs were immobilized
onto different flow cells (Figure 1A).
Alternatively, biotinylated PCR products
(obtained using normal (N-W1282X/NW1282X), heterozygous (N-W1282X/MW1282X) or homozygous (M-W1282X/MW1282X) samples) were immobilized on the
sensor chip (Figure 1B). In both cases, PNA or
DNA probes were then injected.
Sensor chip
The hybridization of 9-mer PNA probes
(Figure 2A and 2B) was compared to that of 9mer DNA probes (Figure 2C and 2D).
Interestingly, the N-W1282X and M-W1282X
PNAs hybridize only to the full complementaBiacore Journal – Number 1 2002
ry target DNAs, the hybridization being much
more efficient than that of the 9-mer DNA probes and the generated PNA:DNA hybrids being
much more stable than DNA:DNA hybrids.
No efficient hybridization occurs between mismatched PNA probes and target W1282X
DNA.
The hybridization between W1282X DNA
or PNA probes and biotinylated PCR products
immobilized on Sensor Chip SA is shown in
Figure 3. The results obtained and described in
detail elsewhere by Feriotto et al. (6) suggest
that W1282X 9-mer PNA probes are able to
generate hybrids, leading to an evident increase
in RUfin and RUres values only in the case of
hybridization between PNA probes and fully
matched target PCR products. Taken together,
these results clearly indicate that the W1282X
9-mer PNAs are efficient probes to generate
hybrids with target PCR products, allowing
real-time identification of the CF W1282X
mutation.
“CF INDEX” FOR SPR-BASED MOLECULAR DIAGNOSIS
We propose the “cystic fibrosis index” (CF
index) as the value (RUfin – RUi)(N)/(RUfin –
RUi)(M), where (RUfin – RUi)(N) are the values obtained with the N-W1282X PNA probe
and the (RUfin – RUi)(M) are those obtained
with the M-W1282X PNA probes (6). The CF
index was found to be usually higher than 4
when PCR products from normal subjects were
employed. The CF index approached 1 when
PCR products from heterozygous W1282X
subjects were employed and lower than 0.4
when PCR products from homozygous
W1282X samples were immobilized on the SA
sensor chip (6).
The results reported by Feriotto et al. (6)
suggest that Biacore’s SPR technology can be
used to detect W1282X mutations in CF, allowing real-time molecular diagnosis of normal,
heterozygous and homozygous W1282X samples. Discrimination between mismatched and
full matched PNA: DNA hybrids is readily and
reproducibly observed during the association
phase conducted under standard Biacore assay
conditions at 25°C in HBS-EP.
2. Feriotto, G., Borgatti, M., Mischiati, C.,
Bianchi, N. and Gambari, R.
J Agr and Food Chemistry 50: 955-62 (2002).
RU
500
400
RUfin
200
RUres
RUi
100
500
1000
Time (s)
B
RU
500
3. Feriotto, G., Lucci, M., Bianchi, N.,
Mischiati, C. and Gambari, R.
Hum Mutat 13: 390-400 (1999).
400
300
4. Feriotto, G., Ferlini, A., Ravani, A.,
Calzolari, E., Mischiati, C., Bianchi, N. and
Gambari, R.
Hum Mutat 18: 70-81 (2001).
5. Nielsen, P.E., Egholm, M., Berg, R. H.
and Buchardt, O.
Science 254: 1497-500 (1991).
200
100
500
C
1000
Time (s)
RU
6. Feriotto, G., Corradini, R., Sforza, S.,
Bianchi, N., Mischiati, C., Marchelli, R. and
Gambari, R.
Lab Invest 81:1415-27 (2001).
200
100
500
Resonance units (RU)
B
RU res
Resonance units (RU)
A
300
References
1. Bianchi, N., Rutigliano, C., Tomassetti,
M., Feriotto, G., Zorzato, F. and Gambari, R.
Clinical and Diagnostic Virology 8: 199-208
(1997).
A
RU fin
SCIENTIFIC REPORT
1000
Time (s)
D
RU
200
Time (s)
100
Resonance units (RU)
C
500
1000
Time (s)
Figure 2
Sensorgrams obtained after injection of
25 µl containing 0.5 µg of normal (dotted
Time (s)
lines) and mutated (solid lines) W1282X
Time (s)
PNA (A, B) and DNA (C, D) 9-mer probes
Figure 3. Sensorgrams obtained after injection of 25 µl containing 0.5 µg of normal (dotted lines) and mutated (solid lines) W1282X 9-mer
to flow cells carrying normal (A, C) or
PNA probes onto flow cells carrying PCR products from normal (A), heterozygous (B) or homozygous (C) W1282X sample. a = injection of the
mutated (B, D) W1282X target DNA (modi-
probes (association); b = injection of HBS-EP (dissociation) (modified from ref. 6).
fied from reference 6).
Biacore Journal – Number 1 2002
PAGE 15