SYBR Green I Analysis of the Trinucleotide Repeat
Responsible for Huntington's Disease
CAMERON N. GUNDRY*, CARL T. WITTWER
Introduction
In 1993 the etiology of Huntington's disease (HD) was revealed as an expansion
of the trinucleotide sequence CAG on chromosome 4pI6.3. Normal individuals
have between 10 and 29 repeats. An intermediate range has been established
between 30 and 35 CAG repeats. Clinical manifestation of the disease occurs with
36-121 repeats, the median being about 44 [Huntington's Disease Collaborative
Reasearch Group, 1993]. Since the causal discovery of the CAG repeat, PCR amplification and subsequent post-PCR gel sizing of the product has been the gold
standard for the diagnosis of HD [Andrew, 1994]. We present a rapid PCR protocol wherein the Till of the PCR product of the CAG repeat region can be used for
Huntington's diagnosis. Melting curve analysis in the presence of SYBR Green I is
used to determine the product T m that is dependent on product length. In addition, we compared melting curves obtained on the ABI 5700 with those obtained
on the LightCycler instrument.
Materials
LightCycler instrument, software and capillaries
Oligonucleotide primers (Operon Technologies, Alameda, CAl
Fluorescein-labeled primers (Operon Technologies, Alameda, CAl
DMSO (Fisher Scientific Company, New Jersey, USA)
LightCycler dNTPs (RMB)
10 mM Magnesium Chloride buffer (Idaho Technology, UT, USA)
KlenTaqTM polymerase (AB Peptides, St. Louis, MO, USA)
Polymerase diluent (Idaho Technology, UT, USA)
SYBR Green I (Molecular Probes, Eugene, OR)
>I- Cameron N. Gundry ( ~ ) (email: Cameron.gundry@path.utah.edu)
Department of Pathology, University of Utah Medical School, Salt Lake City, ur 84132
Equipment
Reagents
Responsible for Huntington's Disease
CAMERON N. GUNDRY*, CARL T. WITTWER
Introduction
In 1993 the etiology of Huntington's disease (HD) was revealed as an expansion
of the trinucleotide sequence CAG on chromosome 4pI6.3. Normal individuals
have between 10 and 29 repeats. An intermediate range has been established
between 30 and 35 CAG repeats. Clinical manifestation of the disease occurs with
36-121 repeats, the median being about 44 [Huntington's Disease Collaborative
Reasearch Group, 1993]. Since the causal discovery of the CAG repeat, PCR amplification and subsequent post-PCR gel sizing of the product has been the gold
standard for the diagnosis of HD [Andrew, 1994]. We present a rapid PCR protocol wherein the Till of the PCR product of the CAG repeat region can be used for
Huntington's diagnosis. Melting curve analysis in the presence of SYBR Green I is
used to determine the product T m that is dependent on product length. In addition, we compared melting curves obtained on the ABI 5700 with those obtained
on the LightCycler instrument.
Materials
LightCycler instrument, software and capillaries
Oligonucleotide primers (Operon Technologies, Alameda, CAl
Fluorescein-labeled primers (Operon Technologies, Alameda, CAl
DMSO (Fisher Scientific Company, New Jersey, USA)
LightCycler dNTPs (RMB)
10 mM Magnesium Chloride buffer (Idaho Technology, UT, USA)
KlenTaqTM polymerase (AB Peptides, St. Louis, MO, USA)
Polymerase diluent (Idaho Technology, UT, USA)
SYBR Green I (Molecular Probes, Eugene, OR)
>I- Cameron N. Gundry ( ~ ) (email: Cameron.gundry@path.utah.edu)
Department of Pathology, University of Utah Medical School, Salt Lake City, ur 84132
Equipment
Reagents