Limitations of Melting Curve Analysis
Using SYBR Green I - Fragment Differentiation
and Mutation Detection in the CFTR-Gene
S. KLEINLE*, K. TABITI AND S. GALLATI
Introduction
Melting curve analysis with the LightCyder instrument can be performed with
the SYBR Green I dye or with fluorescent hybridization probes.
Using the SYBR Green I dye, melting curve analysis offers the opportunity to
identify and characterize PCR products with respect to their melting behavior.
Each double-stranded (ds) DNA product has a specific melting temperature (Tm)
at which 50% of the DNA is single-stranded. Continous monitoring of the denaturation process detects rapid loss of fluorescence near the melting point (T m) and
results in single, sharp melting peaks when plotted as the first negative derivative
of fluorescence versus temperature (-dF/dT) [I].As the Tm of a fragment is a function of the fragment length and the G+C content it can be used e.g. to distinguish
amplification products from short fragments, such as primer dimers.
Using hybridization probes, melting curve analysis monitors the hybridization
of the probes. Melting points of the hybridization probes are also dependent on
probe length and G+C content. Moreover due to their small size, they can detect
small mutations [2,3]. A single base mismatch or small deletions within the probebinding region will decrease the probe's melting point. Hence wildtype, mutant,
and heterozygote samples can be distinguished by different melting points.
We wanted to examine the potential and limitations of fragment differentiation
on melting curve analysis using SYBR Green I dye. We amplified various
sequences of the cystic fibrosis transmembrane conductance regulator (CFTR)gene [4] of increasing length and G+C content, respectively. Our results demonstrate a dear distinction of the different amplification products.
To test limitations of the SYBR Green I dye for the detection of small sequence
differences, such as small mutations, we amplified fragments from the CFTR-gene
comprising different mutations. We analyzed DNA samples from homozygote
wildtype (wt) controls, homozygote mutant patients, and heterozygote patients.
From homozygote wildtype and mutant samples homoduplices are formed on
melting curve analysis while from heterozygote samples heteroduplices between
wildtype and mutant DNA strands arise. We were able to detect 3bp-deletions,
* Stephanie Kleinle (~) (e-mail: skleinle@grnx.de)
Laboratory Dr. Dr. Nevinny-Stickel, Josephspitalstr. 15,80331 Miinchen, Germany
Using SYBR Green I - Fragment Differentiation
and Mutation Detection in the CFTR-Gene
S. KLEINLE*, K. TABITI AND S. GALLATI
Introduction
Melting curve analysis with the LightCyder instrument can be performed with
the SYBR Green I dye or with fluorescent hybridization probes.
Using the SYBR Green I dye, melting curve analysis offers the opportunity to
identify and characterize PCR products with respect to their melting behavior.
Each double-stranded (ds) DNA product has a specific melting temperature (Tm)
at which 50% of the DNA is single-stranded. Continous monitoring of the denaturation process detects rapid loss of fluorescence near the melting point (T m) and
results in single, sharp melting peaks when plotted as the first negative derivative
of fluorescence versus temperature (-dF/dT) [I].As the Tm of a fragment is a function of the fragment length and the G+C content it can be used e.g. to distinguish
amplification products from short fragments, such as primer dimers.
Using hybridization probes, melting curve analysis monitors the hybridization
of the probes. Melting points of the hybridization probes are also dependent on
probe length and G+C content. Moreover due to their small size, they can detect
small mutations [2,3]. A single base mismatch or small deletions within the probebinding region will decrease the probe's melting point. Hence wildtype, mutant,
and heterozygote samples can be distinguished by different melting points.
We wanted to examine the potential and limitations of fragment differentiation
on melting curve analysis using SYBR Green I dye. We amplified various
sequences of the cystic fibrosis transmembrane conductance regulator (CFTR)gene [4] of increasing length and G+C content, respectively. Our results demonstrate a dear distinction of the different amplification products.
To test limitations of the SYBR Green I dye for the detection of small sequence
differences, such as small mutations, we amplified fragments from the CFTR-gene
comprising different mutations. We analyzed DNA samples from homozygote
wildtype (wt) controls, homozygote mutant patients, and heterozygote patients.
From homozygote wildtype and mutant samples homoduplices are formed on
melting curve analysis while from heterozygote samples heteroduplices between
wildtype and mutant DNA strands arise. We were able to detect 3bp-deletions,
* Stephanie Kleinle (~) (e-mail: skleinle@grnx.de)
Laboratory Dr. Dr. Nevinny-Stickel, Josephspitalstr. 15,80331 Miinchen, Germany