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Technical Briefs |
Departments of
1
Biochemistry and
2
Pharmacology, Sultan Qaboos University, College of Medicine, Muscat, Oman
a address correspondence to this author at: Sultan Qaboos University, College of Medicine, Department of Biochemistry, PO Box 35, Al-Khod, Postal code 123, Muscat Sultanate of Oman; fax 968-513-419,
mssimsek{at}omantel.net.om
Single-nucleotide polymorphisms have been detected by various methods, including allele-specific oligonucleotide hybridization (1), allele-specific amplification (2), and restriction fragment length polymorphism (RFLP) analysis (3). In some cases, sequencing of amplified DNA has been used as a direct method (4)(5). RFLP-based methods appear to be superior in genotyping studies. In fact, recently, Cascorbi and Roots (6) recommended the use of RFLP in preference to other methods for single-nucleotide polymorphism detection in the N-acetyltransferase-2 (NAT2) gene. Some groups have used more than one method in their analyses, and when discrepancies occurred between two methods, they used DNA sequencing as a gold standard (7). In this report, we describe a comparison of RFLP methods with automated DNA sequencing for the detection of three different mutations in the NAT2 gene. Our results indicate that caution is needed in the use of automated sequencing to detect heterozygous mutations accurately at some polymorphic sites in the NAT2 gene.
Established PCR-RFLP methods were used to detect NAT2 mutations at the C282T, T341C, and C481T sites. The C-to-T mutations at the 282 and 481 sites were detected with RFLP analysis of a 360-bp DNA fragment with FokI or KpnI digestions, respectively (3). Two different RFLP methods were used for detection of the T341C mutation: a previously described DdeI method (3), and a new complementary NcoI-RFLP. For both methods, a seminested PCR was performed to amplify a 360-bp DNA fragment using a 998-bp DNA fragment as a template. The latter fragment was preamplified as described previously by Hickman and Sim (8), using their Nat-Hu14 and Nat-Hu16 primers. The seminested PCR mixture (50 µL) contained 2 µL of 100-fold diluted 998-bp DNA, 200 µM each dNTP, 1.0 µM primers, 1.25 U of Taq DNA polymerase (Life Technologies), and a buffer consisting of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 1.5 mM MgCl2. Amplification was performed for 30 cycles in a thermocycler (Hybaid Express), with the following conditions for each cycle: 30 s at 95 °C, 1 min at 60 °C, and 30 s at 72 °C. For our NcoI method, the Nat-Hu14 primer (8) was used with a new mutagenesis primer, 341RC (5'-C GAC AAT GTA ATT CCT GCC GTC C-3'). In the DdeI method, the same Nat-Hu14 primer was used together with another modified mutagenesis primer, MS341R (5'-GAC AAT GTA ATT CCT GCC CTC A-3'). The underlined bases in the 341RC and MS341R primers represent mismatched bases with template DNA. RFLP analysis was performed using either 5 U of NcoI (New England Biolabs) or 5 U of DdeI (Life Technologies) in a final buffer volume of 20 µL under mineral oil for 16 h at 37 °C. The resulting fragments were resolved on a 10% polyacrylamide gel. Sequence analysis was performed for 20 DNA samples in a commercial laboratory (Waikato, Hamilton, New Zealand) using a gel-purified 998-bp DNA fragment as a template.
Both DNA chains were sequenced using the Nat-Hu14 primer (8) in the forward sequencing and a new primer, MS 472R (5'-TTC CTC TCT CTT CTG TCA AGCA-3'), for reverse sequencing. The sequencing reactions were performed with an ABI Prism 377 automated DNA sequencer (PE Applied Biosystem) that utilized the BIG DYE terminator chemistry.
Using these automated DNA sequencing and PCR-RFLP methods, we genotyped
20 DNA samples at three polymorphic sites in the NAT2 gene
(C282T, T341C, and
C481T). Table 1
shows a comparison of these genotyping results. The genotypes
determined at the C481T site for all 20 samples
were in complete agreement with the sequencing and KpnI-RFLP
methods. However, the sequencing and RFLP methods yielded different
genotyping results for four samples (Table 1
, samples 8, 9, 10, and
11). These samples were all identified as heterozygous at the
C282T and T341C sites by
the RFLP methods used, but in the sequencing data, all four samples
appeared as homozygous wild type at the T341C
site, and one (sample 8) was identified as a homozygous mutant (T/T) at
the C282T site.
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RFLP analysis of the T341C mutation in some
representative samples using the NcoI method and a
complementary DdeI method is shown in Fig. 1
A. A 360-bp DNA fragment was digested with NcoI or
DdeI, respectively, and the resulting fragments were
separated by electrophoresis on a 10% polyacrylamide gel. A common
fragment, 136 bp (in NcoI) or 150 bp (in DdeI),
was produced in all samples because of the presence of a nonpolymorphic
NcoI or DdeI site within the 360-bp DNA fragment.
In addition to these common fragments, two diagnostic fragments were
produced in both methods: 221 and 198 bp in the NcoI method,
and 211 and 187 bp in the DdeI method. Heterozygous samples
contained two diagnostic fragments, whereas homozygous wild-type
or mutant samples had only one of the diagnostic fragments. One sample
(Fig. 1A
, lane 1/15) was prepared as an artificial heterozygote by
mixing equal amounts of amplified DNA from sample 1 and sample 15. This
artificial mixture was identified correctly as a heterozygote by both
RFLP methods used, but not by automated DNA sequencing.
|
Sequencing results for some representative DNA samples, including the
artificial heterozygous mixture, are shown in Fig. 1B
. The peaks
produced at the T341C site (shown with
arrowheads) were quantified for both the forward and reverse sequencing
reactions (Table 1
). As expected, several heterozygous DNA samples
(Table 1
, samples 4, 7, 12, 14, and 16) all contained two peaks with
good yields at the 341 mutation site, whereas homozygous wild-type or
mutant samples had only one specific peak (e.g., Table 1
, samples 1 and
15). However, the automated sequencing could not identify the
heterozygosity in sample 20, which was an equimolar mixture of samples
1 and 15. A similar problem was observed in the detection of
heterozygotes in other four samples at the T341C
mutation site, and one sample for the C282T site
(Table 1
, samples 8, 9, 10, and 11).
We cannot offer a plausible explanation as to why the detection of heterozygotes was missed in a few DNA samples (4 of 20) by automated DNA sequencing. This may be attributable to inherent problems in the fluorescence-based DNA sequencing, especially for heterozygote detection. One problem is possibly related to the type of DNA polymerase used in the procedure. Leren et al. (4) observed that a G/A heterozygote in the low-density lipoprotein receptor (LDLR) gene was identified correctly by sequenase sequencing, but was missed by sequencing with Taq DNA polymerase. The enzyme used in our case (ABI 377; PE Biosystems) was Ampli-Taq DNA polymerase. Another problem may be attributable to the production of uneven peak-height patterns, which are caused by differences in the efficiency of dideoxy termination at different bases and are affected by sequence context as well. Zakeri et al. (5) reported that a heterozygous C peak was much smaller in size than a heterozygous G peak. This is in agreement with our results. In addition to the production of uneven peaks, a high background, produced by impurities in the DNA template, could further complicate the results, especially for heterozygote detection.
In our case, the background in the electropherograms was fairly low
because all the 998-bp DNA fragments were purified by gel extraction.
The quality of sequence data was actually very good for each sample,
allowing us to read a sequence of 
500 nucleotides, which matched
completely with the published NAT2 gene sequence
(9). However, in a few samples, the signals produced by
heterozygosity at the T341C and
C282T sites were below the set default value
(30%) for heterozygote detection. For example, our quality-control
sample (sample 20) could be scored as a borderline heterozygote in the
reverse sequencing reaction (27% G and 100% A) but not with the
forward primer. If the threshold value of heterozygote detection was
set lower, e.g., 20%, then sample 10 could also be scored as a
heterozygote, at least in one sequencing direction. When a scoring
problem arises for a sample after sequencing in both directions, it
should be considered an undefined genotype until an independent method
is used for its identification. We therefore used independent PCR-RFLP
methods to check the genotypes of all 20 samples at three different
polymorphic sites in the NAT2 gene, and discrepancies were
found between the sequencing and RFLP methods for only four test
samples (Table 1
, samples 8, 9, 10, and 11).
In conclusion, there are some inherent problems in automated DNA sequencing, which may lead to inaccurate heterozygote identification in some samples. Until the associated problems are fully resolved, precautions should be taken in the use of automated sequencing for heterozygote detection. If possible, we recommend the use of two complementary PCR-RFLP methods in this type of analysis.
References
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