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Epicentre Forum 5 (4)
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W. Peng, Endocrinology Department, Royal North Shore Hospital, Sydney,
Australia
Detecting and localizing gene mutations has become an important tool
for genetic disease diagnosis. Conventional methods of detecting point
mutations include single-strand conformation polymorphism (SSCP), denaturing
gradient gel electrophoresis (DGGE), heteroduplex analysis, and ribonuclease
protection assay. However, there are only a few methods such as chain-termination
(Sanger) sequence analysis that can accurately locate a new point mutation.
The Base Excision Sequence Scanning (BESS) T-Scan™ Mutation Detection
and Localization Kit (Epicentre) is a new option for detecting and locating
mutations.1 The BESS T-Scan method
detects all DNA sequence changes involving deoxythymidine. During PCR
amplification, the BESS T-Scan system incorporates a limiting amount
of dUTP in place of dTTP into the PCR product. Treatment of the uracil-containing
PCR product with the BESS T-Scan Excision Enzyme Mix cleaves the sites
of deoxyuridine incorporation, generating nested fragments that are then
separated on a standard sequencing gel.
The BESS T-Scan Kit is a novel alternative to currently used mutation
screening methods. Here, we use the BESS T-Scan Kit with a non-isotopic
label and an ABI 377 DNA Sequencer to detect and locate a single-base
substitution in the untranslated region of exon 7 in the human calcium
sensing receptor gene (CaSR).2 Using
differentially labeled primers, we analyzed three samples in one lane
of a gel. The BESS T-Scan method was rapid and economical. In addition,
it made analysis by SSCP unnecessary.
SSCP analysis
Human genomic DNA was isolated from white blood cells of three individuals
using standard procedures. PCR amplification was performed using a Perkin
Elmer DNA Thermal Cycler. The sequences of the PCR primers used were
T7-6DR: 5'-TAATACGACTCACTATAGGGTCTTCCTCAGAGGAAAGGAGTCTGG-3' (6DR plus
a T7 primer tail) and 6DF: 5'-CAGAAGGTCATCTTTGGCAGCGGCA-3'.1 The 20 µl
amplification reactions contained 0.1 µg of genomic DNA, 2 µl
of GeneAmp® 10X PCR buffer II (Perkin Elmer), 100 mM each dNTP, 0.5 unit
of AmpliTaq Gold™ (Perkin Elmer), and 20 pmoles of each primer.
After an initial denaturation for 10 minutes at 95°C, 45 amplification
cycles were performed as follows: denature for 30 seconds at 95°C,
anneal for 30 seconds at 65°C, and extend for 1 minute at 72°C.
A final extension was performed for 10 minutes at 72°C.
For the single-strand conformational polymorphism (SSCP) analysis, 1 µl
of each PCR product was mixed with 5 µl of formamide. Samples were
heat denatured at 80°C for 1 minute, then chilled to 0°C before
loading onto a 6% native polyacrylamide gel. The gel was subjected to
electrophoresis for 500-700 Vh at 3°C in 1X TBE buffer. After electrophoresis,
the gel was fixed and stained with Silver Stain Plus (Bio-Rad Laboratories).
BESS T-Scan analysis
Dye-labeled T7 primers (JOE, TAMRA, and ROX&$41; were from the T7
Dye Primer Kit (Applied Biosystems Pty Ltd.). The sequence of the T7
primer used was: 5'-TAATACGACTCACTATAGGG-3'. The BESS amplification reactions
using the dye (TAMRA)T7-6DF primer set and the dye (ROX)T7-6DF primer
set were performed in a 10 µl volume. Each reaction contained 0.2
unit of Taq DNA polymerase, 1X PCR buffer, 1.5 mM MgCl2, 0.8 µl
of dye-labeled T7 primer, 0.8 pmole of 6DF primer, 0.8 µl of BESS
T-Scan dNTP Mix, and 1 µl of PCR product (from above, diluted 1:100).
A 5 µl amplification reaction was used with the dye(JOE)T7-6DF
primer set and the volume of each component was adjusted accordingly.
Thermal cycling conditions were 25 cycles of 95°C for 30 seconds,
55°C for 30 seconds, and 72°C for 1 minute. A final extension
was performed for 10 minutes at 72°C. After amplification, the 3
sets of PCR products were combined (total volume was 25 µl). Then
6 µl of 10X BESS T-Scan Excision Enzyme Buffer and 3 µl of
BESS T-Scan Excision Enzyme Mix were added. Deionized water was added
to a final volume of 60 µl and the reaction was incubated for 30
minutes at 37°C. The DNA was then precipitated by adding 3 µl
of sodium acetate (pH 5.3) and 180 µl of 100% ethanol. The DNA
was pelleted by centrifugation for 15 minutes, the supernatant was discarded,
and the pellet was dried. Then the samples were separated by electrophoresis
on an ABI 377 DNA Sequencer (electrophoresis was performed at SUPAMAC,
Sydney University and Prince Alfred Macromolecular Analysis Centre).
The SSCP analysis of the untranslated region of exon 7 of the human
CaSR gene is presented in Figure 1. Lane 2 shows the polymorphic banding
pattern of one individual, predicting a heterozygous mutation in this
463 bp fragment, but without accurate localization. Note that the gel
pattern for the heterozygous mutant is almost the same as the pattern
of the normal samples.
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Figure 1. SSCP analysis of the untranslated region in exon 7
of the human CaSR gene. SSCP analysis was performed as described
in the text. Samples 1 and 3, wild-type banding patterns; Sample
2, human CaSR gene showing a polymorphic banding pattern. |
Figure 2 shows the BESS T-Scan analysis. The samples used in this analysis
were the same as those used in the SSCP analysis. The electropherogram
showing the three samples in one lane was converted to individual electropherograms
making the comparisons more convenient. The analysis of sample 2 showed
an additional peak at position 61 of the electropherogram, meaning one
base was converted to an extra T in the reverse sequence. This analysis
confirmed the heterozygous polymorphism that was predicted in the SSCP
analysis. Also, this BESS T-Scan analysis located the single-base substitution
(T–>A) at base #3751 of the human CaSR gene.3
 |
Figure 2. BESS T-Scan analysis of the untranslated region in
exon 7 of the human CaSR gene. BESS T-Scan reactions
were performed as described in the text and the samples separated
on an ABI 377 DNA Sequencer. The arrow indicates the site of mutation. |
The BESS T-Scan method directly detected and localized a single-base
substitution in the human CaSR gene making SSCP scanning unnecessary.
The method introduced here combines advantages of both the BESS T-Scan
Kit and the ABI DNA Sequencer. It was non-isotopic, rapid, economical,
and reliable, and can be used as an alternative to DNA sequencing for
routine genetic disease diagnoses.
- Hawkins, G.A. et al. (1997) Epicentre Forum 4 (2),
1.
- Pollak, M.R. et al. (1993) Cell 75, 1297.
- Garrett, J.E. et al. (1995) GenBank HSU20760.
| BESS MutaScan™ Mutation Characterization Kit |
BESS-T&G™ Base Reader Kit |
| BESS T-Scan™ Mutation Detection and Localization Kit |
BESS-T™ Base Reader Kit |
| BESS G-Tracker™ Mutation Detection and Localization Kit |
BESS-G™ Base Reader Kit |
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