Rapid Genotyping of 2-bp and 9-bp Deletion Mutations
Using the LightCycier Instrument
TSUTOMU AOSHIMA, MITSUHARU KAJITA, YOSHITAKA SEKIDO, SHUNJI MIMURA,
KAZUYOSHI WATANABE, KAORU SHIMOKATA, TOSHIMITSU NIWA"
Introduction
Instead of sequencing the candidate gene PCR fragment, simple methods such
as restriction enzyme digestion after PCR are used to detect known mutation.
In some cases, however, such methods cannot be applied and sometimes lead to
an ambiguous result. A faster method would be required in case of mass screening or emergency. Recently, an elegant method to detect a known mutation with
the LightCycler has been introduced. Rapid real-time PCR is monitored by fluorescent oligonucleotide probes that hybridize the target region of the PCR
product. Only when the probes hybridize the template fluorescence resonance
energy transfer occurs thereby producing a specific fluorescence emission.
Then, by slowly heating the PCR product in the probe-hybridized state, the
diminishing fluorescence value gives a melting curve. In the presence of mismatch sequences between the template and the probe, the probe melts off at a
lower temperature. Therefore, the melting curve clearly demonstrates its genotype as the differences in T m' This fluorescence PCR on the LightCycler has displayed its marked ability to detect single nucleotide substitution [1,2]. However, many inherited diseases are caused by small deletion mutations as well as
single nucleotide mutations.
In this report, we present the methods for rapid genotyping of such small
deletion mutations. First, using a probe-based hybridization method, we detected a 2-bp deletion mutation in the DNA of a patient with Fabry disease, which is
an X-linked recessive disorder caused by the deficient activity of a-galactosidase
(a-Gal;EC 3.2.1.22). We were able to clearly genotype his family. Next, using an
SYBR Green I-based method without hybridization probes, we detected a 9-bp
deletion mutation in the cDNA of a patient with carbamoylphosphate synthetase
I (CPS1; EC 6.3.4.16) deficiency, which is an autosomal recessive disorder affecting the first enzyme step of the urea cycle [3].
* Toshimitsu Niwa (~) (e-mail: tniwa@med.nagoya-u.ac.jp)
Department of Clinical Preventive Medicine, Nagoya University, School of Medicine,
65 Tsuruma-Cho, Showa-ku, Nagoya 466-8550, Japan
Using the LightCycier Instrument
TSUTOMU AOSHIMA, MITSUHARU KAJITA, YOSHITAKA SEKIDO, SHUNJI MIMURA,
KAZUYOSHI WATANABE, KAORU SHIMOKATA, TOSHIMITSU NIWA"
Introduction
Instead of sequencing the candidate gene PCR fragment, simple methods such
as restriction enzyme digestion after PCR are used to detect known mutation.
In some cases, however, such methods cannot be applied and sometimes lead to
an ambiguous result. A faster method would be required in case of mass screening or emergency. Recently, an elegant method to detect a known mutation with
the LightCycler has been introduced. Rapid real-time PCR is monitored by fluorescent oligonucleotide probes that hybridize the target region of the PCR
product. Only when the probes hybridize the template fluorescence resonance
energy transfer occurs thereby producing a specific fluorescence emission.
Then, by slowly heating the PCR product in the probe-hybridized state, the
diminishing fluorescence value gives a melting curve. In the presence of mismatch sequences between the template and the probe, the probe melts off at a
lower temperature. Therefore, the melting curve clearly demonstrates its genotype as the differences in T m' This fluorescence PCR on the LightCycler has displayed its marked ability to detect single nucleotide substitution [1,2]. However, many inherited diseases are caused by small deletion mutations as well as
single nucleotide mutations.
In this report, we present the methods for rapid genotyping of such small
deletion mutations. First, using a probe-based hybridization method, we detected a 2-bp deletion mutation in the DNA of a patient with Fabry disease, which is
an X-linked recessive disorder caused by the deficient activity of a-galactosidase
(a-Gal;EC 3.2.1.22). We were able to clearly genotype his family. Next, using an
SYBR Green I-based method without hybridization probes, we detected a 9-bp
deletion mutation in the cDNA of a patient with carbamoylphosphate synthetase
I (CPS1; EC 6.3.4.16) deficiency, which is an autosomal recessive disorder affecting the first enzyme step of the urea cycle [3].
* Toshimitsu Niwa (~) (e-mail: tniwa@med.nagoya-u.ac.jp)
Department of Clinical Preventive Medicine, Nagoya University, School of Medicine,
65 Tsuruma-Cho, Showa-ku, Nagoya 466-8550, Japan