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1
Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD 20852.
a Address correspondence to this author at: 12420 Parklawn Dr., Suite 451, MSC 8110, Rockville, MD 20852. Fax 301-443-8579; e-mail
rlipsky{at}mail.nih.gov.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We used a double-stranded DNA-specific fluorescent dye, SYBR Green I (SYBR) in an efficient system (PE 7700 Sequence Detector) in which DNA melting was controlled and monitored in a 96-well plate format. We measured the decrease in fluorescence intensity that accompanied DNA duplex denaturation, evaluating the effects of fragment length, dye concentration, DNA concentration, and sequence context using four naturally occurring polymorphisms (three SNPs and a single-base deletion/insertion).
Results: DNA melting analysis (DM) was used successfully for variant detection, and we also discovered two previously unknown SNPs by this approach. Concentrations of DNA amplicons were readily monitored by SYBR fluorescence, and DNA amplicon concentrations were highly reproducible, with a CV of 2.6%. We readily detected differences in the melting temperature between homoduplex and heteroduplex fragments 15–167 bp in length and differing by only a single nucleotide substitution.
Conclusions: The efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 per 500 nucleotides in coding
sequences and at a higher rate in noncoding sequences
(1). Genome wide there are several million common SNPs. SNPs
are highly amenable for high-throughput genotyping using efficient
methodologies, including DNA arrays (2)(3)(4), mass
spectrometry (5), and PCR end-plate read methods such as
TaqMan (PE 7700 Sequence Detector) (6). For this reason,
detection of SNPs of moderate or high abundance (i.e., with rare allele
frequencies of >10%) has been made a high priority and may be
successfully accomplished by analyzing a relatively small complement of
chromosomes. Sequencing of 10 chromosomes (five persons) detects
approximately two-thirds of all sequence variants with a frequency of
10%. However, SNPs that alter gene expression or affect structure of
the gene product often are rare. Sequencing of 10 chromosomes detects
<10% of sequence variants with a frequency of 1%, a frequently used
definition for polymorphism. The continuing requirement for detection of rare SNPs will maintain the need for high-throughput, inexpensive methods for sequence variant detection. Sequence variant detection methods access physical properties of DNA. Several of the most powerful methods for SNP detection [e.g., denaturing gradient gel electrophoresis (DGGE) and denaturing HPLC (dHPLC)] are ultimately based on the thermodynamic properties of DNA duplexes or single-stranded DNA [i.e., single strand conformational polymorphism (SSCP) analysis].
For many years it has been understood that the relative thermostability of a DNA duplex increases with increasing GC content. Subsequently, it has become apparent that DNA duplex stability is also dependent on additional factors. Mathematical models have predicted and empirical results verified that the largest contribution by far to DNA helix stability actually originates from the vertical stacking of base pairs (7) and that the stability of double-stranded DNA (dsDNA) depends largely on the identities of the nearest-neighbor bases that determine this stacking (7)(8). For oligonucleotide sequences in solution, a two-state (i.e., duplex and random coil) nearest-neighbor model reliably predicts the stability of DNA duplexes with Watson-Crick pairs (9)(10). Sequence composition-dependent differences in homoduplex stability were first exploited by Lerman and Silverstein (11), who detected sequence variations of DNA by use of DGGE.
The nearest-neighbor model was extended for heteroduplex stability to
include parameters for interactions between the mismatches and
neighboring bases (10)(12)(13).
Allawi and SantaLucia (10) found good agreement between
predicted and observed thermodynamic parameters
(
G37, H, and
S) of 45 single internal G·T mismatched
oligonucleotides of different base composition and length. In addition,
the magnitude of the difference in the melting temperature
(Tm) of single-base mismatches vs
single-base substitutions in homoduplexes was large in these
oligonucleotides (i.e., 2–5 °C). The nearest-neighbor model also
predicts the stability and thermodynamics of DNAs containing other
internal single-base mismatches (C·T, A·C, G·A)
(14)(15)(16). A difference in the thermodynamic stability of
heteroduplex DNA directly explains the improved efficiency of gel-based
methods such as DGGE to detect sequence variations by comparison of a
heteroduplex DNA with component homoduplex DNAs. This contrasts with
the reduced sensitivity obtained by comparing a homoduplex DNA with the
homoduplex DNA containing a single nucleotide substitution that is
correctly Watson-Crick base paired.
Our experimental approach was to use heteroduplex melting in solution to detect single nucleotide sequence variations. The goal was to create a high-throughput system for the detection of sequence variants within polynucleotide PCR amplicons. Two enabling elements for this new approach are SYBR Green I (SYBR), a dye that fluoresces when bound to dsDNA, and the PE 7700 Sequence Detector, which enables simultaneous DNA melting and fluorescence quantification in a 96-well format. In this study, we used three naturally occurring SNP variants and a single nucleotide insertion/deletion to explore the effects of dye concentration, DNA concentration, and fragment length on the ability to detect SNPs by heteroduplex melting in solution by DNA melting analysis (DMA).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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pcr amplification of human genomic dna
DNA fragments of 60 bp or greater were synthesized by PCR on
genomic DNA templates. For DMA experiments using known SNPs, such as
HTR2A 102T
C, we selected DNAs from individuals who were
either homozygous for an allele (HTR2A 102TC 102T/102T)
or heterozygous (HTR2A 102T/102C). The total volume for the
PCR reaction was 25 µL and contained 100 ng of genomic DNA, 0.25 mM
dNTPs, 0.5 µM PCR primers, and 0.75 U of AmpliTaq Gold with
appropriate buffer (Perkin-Elmer). PCR master mixtures were prepared
daily. The denaturation and extension steps for all PCRs were at
95 °C for 15 s and 72 °C for 30 s in 30 cycles,
respectively. The annealing temperature for the 60-, 78-, 100-, and
152-bp amplicons was 50 °C for 20 s. Each fragment was
amplified using the following primers:
For other known SNPs, the annealing step for the variants tested
was 20 s. Annealing temperatures were 55 °C for
HTR2A His452Tyr, 54 °C for COMT Val158Met, and
59 °C for DRD2-141C ins/del. For screening of
unknown SNPs, the annealing step was performed at 60 °C for 20
s. PCR amplicons used for SNP screening by DMA were based on exon
sequences of the
N-methyl-D-aspartate receptor
gene (NR1; GenBank Accession No. Z32773) and both exon and
intron sequences from the acetylcholine receptor gene (AChR)
-subunit (GenBank Accession No. X02502). The primer sequences used
to amplify these sequences were as follows:
Following PCR amplification, each product was purified using either the Qiagen reagent set for PCR or gel extraction. The purified product was then denatured at 95 °C for 4 min and reannealed by slowly cooling to 60 °C over a period of 30 min to permit the formation of a mixture of homoduplex and heteroduplex molecules in the DNA amplified from heterozygous individuals.
sybr fluorescence detection of dna melting
The double strand-specific dye, SYBR Green I (SYBR), was obtained
from Molecular Probes and used for DMA. The optimal excitation and
emission spectra of SYBR are centered at 492 and 513 nm, respectively.
SYBR is supplied as a 10 000x concentrate by the
manufacturer, with no molar concentration values or formula
weights being supplied. The optimal concentration of SYBR used in our
experiments was 3.6x dye (reduced from 10 000x), and dilutions were
with 1x Tris-borate-EDTA buffer. The 3.6x SYBR dye concentration was
optimal for concentrations of DNA between 20 and 100 ng in 15 µL.
Fluorescence measurements and denaturation were accomplished using the
PE 7700 Sequence Detector. Fluorescence signals were recorded
approximately every 7 s over the entire time course of
denaturation, which varied from 30 min to 4 h, gathering data for
up to 96 samples at a time.
data analysis
Following DNA duplex denaturation and data acquisition, raw
fluorescence data were exported to Microsoft Excel for statistical
analyses. Fluorescence data from melting curves were converted into
Tm by plotting the negative derivative
of fluorescence vs temperature (-dF/dT vs
T). Melting point predictions were performed using MeltCalc
software (17).
variant screening using dHPLC
Following PCR amplification, samples were denatured and reannealed
as described above for DMA to enhance formation of DNA heteroduplexes.
Samples were then processed using a Transgenomic dHPLC, consisting of a
96-well autosampler, column oven, pumps, degasser, variable wavelength
ultraviolet detector, sample loop, and a PC-based data
collection system. Before dHPLC, melting curves for PCR amplicons were
simulated using the Transgenomic WavemakerTM
software to determine whether any significant shifts in
Tm could be predicted for the
amplicon. A Transgenomic DNASep column was used for separations.
Buffers used on the column were as follows: buffer A, consisting of 10
mmol/L triethylammonium acetate (pH 7.4); and buffer B, consisting of
10 mmol/L triethylammonium acetate containing 250 mL/L
acetonitrile. Loading buffer consisted of 80 mL/L acetonitrile.
Tms and buffer gradients were
determined using the Transgenomic melting temperature predictions
software.
dna sequence analysis of candidate SNPs
Genomic DNA samples that were determined by DMA to contain a SNP
were amplified using PCR, and the products were purified as described
above to eliminate excess primer and genomic DNA. The sequencing
reaction was performed in a 10-µL reaction containing 10 nmol/L one
primer (forward or reverse), 4 µL of BigDyeTM
Terminator Cycle Sequencing reaction mixture (Perkin-Elmer), and 3 µL
of the purified PCR product. The temperature cycle for the sequencing
reaction consisted of 25 cycles of 10 s at 96 °C, 5 s at
56 °C, and 4 min at 60 °C. Purification of this reaction was
performed using a gel filtration block (AGTC). The purified reaction
products were dried under reduced pressure and resuspended in
formamide and sequencing dye mixture. Products were resolved on an ABI
377 automated sequencer. The determined sequence was aligned and
analyzed using the ABI AutoAssembler software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C; GenBank Accession No. X57830). These analyses were
actually performed by comparing the melting characteristics of the DNA
homoduplex to a mixture of homo- and heteroduplexes that naturally
result from the amplification or synthesis of dsDNA and its
denaturation and slow reannealing. As shown in Fig. 1
, amplification, denaturation, and reannealing of heterozygous
DNA will yield four dsDNA duplexes. The two homoduplexes constitute at
least 50% of the reannealed dsDNA, and the two heteroduplex DNAs
constitute at most 50% of the reannealed dsDNA. It is important to
recognize that each one of the four dsDNA species formed during
reannealing has different thermodynamic characteristics. For small DNA
duplexes, G·T mismatches are less thermodynamically destabilizing
than A·C mismatches (10). However, the magnitude of the
difference in melting thermodynamics of the two heteroduplex DNA
species makes them readily distinguishable from either of the
homoduplex dsDNAs.
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An important limitation of our approach is that both the structure and concentration of SYBR are proprietary. SYBR is reported to bind to the minor groove of dsDNA (Molecular Probes Manual). Once bound and excited at a wavelength of 450 nm, it has a maximum emission signal at 513 nm.
concentration of dna and sybr
Increasing the DNA concentration increases the
Tm of duplex DNA in solution.
Therefore, the effect of varying the DNA concentration and the ability
to control for variation in DNA concentration were critical to our
ability to detect sequence variation by DMA. dsDNA concentrations could
vary as a result of different efficiencies in DNA amplification.
To approach this issue, the extent of variation in initial dsDNA
concentrations before melting was monitored. For these determinations,
we used the HTR2A 100-bp amplicon. As shown in Fig. 2A
, the DNA concentration could be measured on the ABI 7700 by
monitoring SYBR fluorescence intensity, because this intensity is
linearly proportional to dsDNA concentration. It was noted that higher
concentrations of SYBR quenched the fluorescence signal (Fig. 2B
) and
that the relationship between SYBR and this quenching was again linear.
Quenching by excess SYBR may be attributable to the presence of unbound
SYBR. Therefore, it is important to use a constant concentration of
SYBR if DNA concentrations are to be determined.
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dsDNA quantification revealed that the concentration of a particular DNA amplicon was highly reproducible as determined by the SYBR fluorescence signal. The within-day imprecision (CV) for the HTR2A 100-bp amplicon from genomic DNA from an individual heterozygous for the T102C variant was 2.6% at a mean concentration of 1.98 g/L. Thus, amplicon concentrations were highly reproducible. Similar results were also obtained for different amplicons using different primer combinations (data not shown). Therefore, DNA concentrations could be reliably monitored in all of the samples before DMA, using the same device in which DMA was conducted, namely the ABI 7700.
Increasing the dsDNA concentration increased the Tm of the 100-bp HTR2A amplicon. In the presence of SYBR, as DNA concentration was increased from 0.5 mg/L to 10 mg/L, the melting curves were shifted to the right, as expected (data not shown).
The effect of different concentrations of SYBR on the temperature of
dsDNA denaturation was evaluated (Fig. 2C
) using the HTR2A
100-bp amplicon at a concentration of 2 mg/L in a 15-µL final assay
volume. This was a convenient DNA concentration given that the typical
yield of a PCR reaction was 10 mg/L in a volume of 30 µL. The
manufacturer recommends that SYBR, supplied as a stock, be used after
diluting 1:10 000. We determined that the optimal dye concentration
for a wide range of DNA concentrations was 3.6x. The use of lower SYBR
concentrations led to low emission signals, but higher dye
concentrations caused signal quenching. In addition, increasing the
concentration of SYBR from 1.8x to 57.6x shifted the DNA melting
curve to the right, indicating that by binding the dsDNA, the dye is
stabilizing the DNA duplex, thereby increasing the temperature at which
it is denatured. In fact, at higher SYBR concentrations relative to a
constant DNA concentration, it could be seen that the dsDNA did not
completely denature, as evidenced by the melting curves obtained using
28.8x and 57.6x SYBR (Fig. 2C
), which are shifted far to the right.
ability to detect single-base mismatches in dna fragments of
different lengths
Six dsDNA fragments of different lengths were either synthesized
(15 and 25 bp) or PCR amplified (60, 80, 100, and 152 bp). All
fragments had the same TC polymorphism located in the middle of the
same target HTR2A gene sequence. The effect of the
single-base pair mismatches (G·T and A·C) was assessed by comparing
the thermodynamic stability of the DNA heteroduplexes with homoduplex
DNA (A·T). A 97-bp fragment was also amplified in which the mismatch
was located 30 bp from the 5' end (see Table 1
).
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When the 15- or 25-bp dsDNA fragment was subjected to a temperature
increment of 2 °C/min, a readily observable difference in the rate
of decrease in SYBR fluorescence was observed for homoduplex dsDNA
compared with the heteroduplex/homoduplex DNA mixture. The fluorescence
data from each melting curve were converted into the
Tm by plotting the negative
derivative of fluorescence vs temperature (-dF/dT vs
T). A representative derivative plot for the 15-bp fragment
is shown in Fig. 3A
. The Tm between the
homoduplex DNA and the heteroduplex/homoduplex mixture was 5 °C
for the 15-bp fragment and 3.5 °C for the 25-bp fragment (Fig. 3A
and Table 1
). Each observed value was within the range of
Tm values predicted for that
fragment (Table 1
).
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For longer dsDNAs, the rate of DNA melting was decreased to
0.5 °C/min for the 60- and 80-bp fragments and to 0.067 °C/min
for fragments 97–152 bp in size. When this was done, the differences
in the melting rates between homoduplex dsDNA and the
heteroduplex/homoduplex mixture were again readily distinguishable. The
observed Tm for these fragments
varied between 0.9 °C (152 bp) and 5.5 °C (60 bp; Fig. 3, B–D
,
and Table 1
).
detection of additional sequence variants using dma
Two additional SNP variants and a single nucleotide
insertion/deletion were also examined by DMA using amplicons 94, 110,
and 133 bp in size. The SNPs, both of which produce amino acid
substitutions, were HTR2A 1499CT (His452Tyr; GenBank
Accession No. X57830) and COMT 1947GA (Val158Met; GenBank
Accession No. Z26491) with fragment sizes of 94 and 110 bp,
respectively. The insertion/deletion used was
DRD2-141Cins/del (GenBank Accession No. X53502) in a 133-bp
fragment (Fig. 4C
). In all of these cases, the homoduplex/heteroduplex mixture
produced derivative melting profiles that were distinguishable from the
melting profiles for the homoduplex (Fig. 4
). The
homoduplex/heteroduplex mixture always had a lower
Tm than that for the homoduplex (Fig. 4
). The observed Tm for each
amplicon heteroduplex was 1.2–3.8 °C (Table 1
).
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snp discovery using dma
The previous experiments allowed us to evaluate the merits of DMA
using known SNPs. We wanted to use DMA to discover new SNPs and to
validate our approach using previously known methods for SNP discovery
and identification, namely dHPLC and direct DNA sequence analysis of
PCR amplicons. To approach the issue of validation, we selected
amplicon sizes that could also be easily screened by dHPLC. The PCR
amplicons produced for these experiments were derived from portions of
two different genes: (a) exon sequences of the
NR1 gene, and (b) exon and intron sequences from
the AChR gene (see Materials and Methods). The
two PCR amplicons were 163 bp (NR1) and 167 bp
(AChR) in size and were amplified from genomic DNA prepared
from six unrelated individuals. The PCR products were thermally
denatured and allowed to reanneal, after which DMA was performed. The
melting profile of each sample was followed, and the fluorescence data
from melting curves were then converted into the
Tm using the derivative melting
profile (-dF/dT vs T). Derivative
melting data for representative NR1 and AChR PCR
amplicons are presented in Figs. 5A
and 6A
. One sample from the NR1 gene amplification and one
sample from the AChR gene amplification, each originating
from a different individual, showed a shift in
Tm when compared with the melting
peaks obtained for the other NR1 gene or AChR
gene-derived samples. These results suggested the presence of
heteroduplex/homoduplex DNAs. These melting profiles were repeated with
similar results. The observed Tms
for these fragments were 2.2 °C (163-bp NR1) and
0.6 °C (167-bp AChR; Table 1
).
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The two genomic DNA samples that produced the
Tm shifts were then independently
amplified for DNA sequence analysis. Direct DNA sequence analysis of
these PCR products showed the presence of a SNP within the amplicon
(Fig. 7
). No other variants were detected in either of these PCR
products. Direct sequencing of the NR1 or AChR
PCR products not showing a Tm shift
indicated that they did not contain a SNP within the amplicon
(data not shown). In addition, we confirmed that the new SNPs
discovered in the NR1 and AChR genes by DMA could
be replicated using dHPLC (data not shown). It should be noted that
each SNP was located in a region of the PCR amplicon that was predicted
to behave as a single melting domain (Figs. 5B
and 6B
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In principle, all dsDNA heteroduplex mismatches in amplicons 100 bp or
less in size may be detectable by DMA. Our data showed that differences
in melting profiles for homoduplex-heteroduplex DNA mixtures containing
fragments 15–100 bp in length with single A·C and G·T internal
mismatches were readily detectable by DMA. The observed
Tm for these different fragments
varied between 1.4 and 5.5 °C (Table 1
). For shorter DNA fragments
containing either single A·C or G·T mismatches, A·C mismatches
were the least stable thermodynamically, whereas G·T mismatches were
the most stable (15). Thus, DMA may allow direct access to
entropy and enthalpy changes that result from single Watson-Crick
mismatches. It should be noted that internal single-base mismatches
could produce melting instability in larger fragments (>100 bp) and
that our method was sufficiently sensitive to detect a melting
difference in a homoduplex-heteroduplex mixture that contained either a
single internal A·G mismatch or a single internal C·T mismatch (the
AChR gene-derived fragment; Table 1
).
Conceptually, DMA is simple. It quantifies the difference in heteroduplex stability while avoiding intervening steps, such as gels and columns, whose effectiveness depends on principles other than the thermodynamic properties of DNA. The conceptual simplicity of DMA leads to practical advantages over other high-throughput SNP detection methods. In SSCP, the SNP may not alter internal base pairing. In SSCP analysis, gel mobility is dependent on fragment size, the complex tertiary structures formed, and the way these conformations interact with the gel matrix. Different conformations may therefore have the same SSCP mobility. The lack of sensitivity of SSCP analysis led to a series of other SSCP approaches (e.g., the use of special gel constituents and SSCP analysis of multiple fragments containing the same sequence). The increased labor involved in these methods improves the sensitivity of SSCP analysis but also underlines its limitations. dHPLC is a relatively high-throughput method that is well suited to automation. However, as with SSCP analysis, the electrophoretic behavior of the DNA fragments can be erratic because we cannot yet predict the structure of the partially melted DNAs, nor can we predict their interaction with the immobile phase (i.e., reversed-phase columns). In dHPLC, the mobility of the partially melted DNA structure is highly dependent on the exact temperature and other conditions (e.g., pH or acetonitrile gradient) chosen.
DGGE most closely resembles DMA because the variant is detected based on the way in which the substitution alters the thermodynamic properties of heteroduplex or substituted homoduplex dsDNA. Like dHPLC and SSCP analysis, however, DGGE requires a shift in gel mobility, which depends on many other variables. Finally, chemical and enzymatic mismatch cleavage both depend on molecular recognition of a mismatch site.
An understanding of the effects of sequence context on DNA melting has
been largely developed using DNA oligomers <25 bp in length. For
oligomeric DNAs, the thermodynamic parameters
Tm, G, H,
and S can be estimated using the output calculated by the
MeltCalc program (17), which is based on estimates of
nearest-neighbor parameters for dsDNA with or without mismatches
as described by Allawi and SantaLucia
(10)(14)(15)(16). However, the nearest-neighbor
model has limitations because the melting of oligomers
frequently, but not always, fits a two-state model (i.e., the DNA
exists as either random coil or double helix). With longer DNA
fragments, such as those used in our studies, this two-state model
cannot be used because longer DNA fragments can have other unknown
secondary structures, such as hairpin turns and loops. Water molecules
and ions can also affect the relative stability of these structures
(18). The results from Table 1
support the idea that some
deviations between mathematically predicted values and observed values
of Tm are also attributable to
different structural characteristics of the longer DNA fragments.
In DMA, SYBR stabilizes DNA duplexes, and as illustrated in Fig. 2
, doubling the dye concentration increased the
Tm by 1–2 °C over large a range of
dye concentrations. It is well known that both the DNA concentration
and the time allowed for annealing or melting are critical factors for
denaturation (19). Our results show that DMA can be
successfully used for variant detection provided that three factors,
DNA concentration, transition time, and SYBR concentration, are all
carefully controlled. In practice, only one of these three factors,
i.e., DNA concentration, is likely to vary from one sample to the next.
A low dsDNA concentration could simulate the melting behavior of
heteroduplex DNA. Fortunately, our DNA yields were highly consistent
because the amplifications were carried out consistently and under
endpoint conditions, i.e., to >30 cycles, starting with >10 ng of
genomic DNA template. These PCR conditions are fairly typical.
Moreover, with DMA it is easy to detect significant amounts of
variation in the initial dsDNA concentration using the initial
fluorescence intensity as a quantitative measure.
The PE 7700 Sequence Detector (ABI Prism) was used to monitor the
decrease in SYBR fluorescence signal over the course of dsDNA melting.
With this particular instrument, it is possible to perform denaturation
experiments and analyze the DNA melting curves in 96 samples at a time.
The detector monitors the emission fluorescence signal approximately
every 7 s, providing a very detailed and descriptive curve of DNA
duplex melting behavior during the course of denaturation. With these
detailed DNA melting curves, Watson-Crick mismatches in dsDNAs
100–150 bp in size were readily detected, with an observed
Tm of 1–5 °C.
Recently, melting analysis has been used for genotyping. Marziliano et
al. (20) used a Tm assay to
detect differences in the melting profile of a 132-bp PCR product
having a TA insertion in the TATA box of the UGT1A gene
promoter compared with the nonvariant reference promoter sequence. The
melting profiles of the PCR products from individuals that were
homozygous for either the deletion or the nonvariant sequence
(reannealed amplicons composed of homoduplex DNA) were readily
distinguishable from PCR products produced from heterozygous
individuals (reannealed amplicons composed of heteroduplex DNA). It
should be noted that the derivative melting profiles of the homoduplex
samples and the heteroduplex samples generated single
Tm peaks, similar to our own melting
profiles (Figs. 5A
and 6A
).
In a separate study using melting curve analysis with a
LightCyclerTM (Roche Molecular Biochemicals),
Aoshima et al. (21) detected differences in melting profiles
between small PCR amplicons of 46 or 55 bp, the former having a
internal 9-bp deletion, derived from a CPS1 gene cDNA.
However, the authors expected that the
Tm of a heteroduplex containing the
9-bp deletion and a homoduplex derived from a 100-bp fragment
(Tm = 0.6 °C) would make the
melting difference undetectable with SYBR. However, we successfully
detected SNPs in fragments >100 bp, including an unknown SNP in the
AChR gene located within a 167-bp amplicon. For the
AChR gene amplicon, the ability of DMA to detect a small
melting temperature shift (observed
Tm = 0.6 °C) may have been aided
by the location of the SNP, which was near a region of decreased
stability (lower GC content; see Fig. 6B
). In addition, we used a
slower heating rate for larger amplicons than that used by Aoshima et
al. (21). Although additional comparative studies are
certainly warranted to fully describe the power of DMA, the efficiency
and sensitivity of this approach, as implemented on the PE 7700
Sequence Detector (and potentially the ABI 5700 and ABI 7900 HT), makes
it highly suitable for large-scale detection of new sequence variants.
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Footnotes |
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2 These authors contributed equally to the project.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L. L.M. Poon, O. K. Wong, W. Luk, K. Y. Yuen, J. S.M. Peiris, and Y. Guan Rapid Diagnosis of a Coronavirus Associated with Severe Acute Respiratory Syndrome (SARS) Clin. Chem., June 1, 2003; 49(6): 953 - 955. [Full Text] [PDF]
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 R. J. Wurzburger, R. Gupta, A. P. Parnassa, S. Jain, J. A. Wexler, J. L. Chu, K. B. Elkon, and R. D. Blank Use of GC Clamps in DHPLC Mutation Scanning Clin. Med. Res., April 1, 2003; 1(2): 111 - 118. [Abstract] [Full Text] [PDF]
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N. von Ahsen Labeled Primers for Mutation Scanning: Making Diagnostic Use of the Nucleobase Quenching Effect Clin. Chem., March 1, 2003; 49(3): 355 - 356. [Full Text] [PDF]
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C. N. Gundry, J. G. Vandersteen, G. H. Reed, R. J. Pryor, J. Chen, and C. T. Wittwer Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes Clin. Chem., March 1, 2003; 49(3): 396 - 406. [Abstract] [Full Text] [PDF]
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