High-resolution genotyping by amplicon melting analysis using LCGreen.

BACKGROUND High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. METHODS We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. RESULTS The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. CONCLUSIONS High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

[1]  Carl T. Wittwer,et al.  Rapid Cycle Real-Time PCR — Methods and Applications , 2002, Springer Berlin Heidelberg.

[2]  D. Poland,et al.  Recursion relation generation of probability profiles for specific‐sequence macromolecules with long‐range correlations , 1974, Biopolymers.

[3]  C. N. Gundry,et al.  Real-time multiplex PCR assays. , 2001, Methods.

[4]  Kirk M. Ririe,et al.  Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. , 1997, Analytical biochemistry.

[5]  C. N. Gundry,et al.  Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes. , 2003, Clinical chemistry.

[6]  U J Balis,et al.  The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. , 1997, BioTechniques.

[7]  Robert Lipsky,et al.  DNA melting analysis for detection of single nucleotide polymorphisms. , 2001, Clinical chemistry.

[8]  Stephen A. Bustin,et al.  Real-Time PCR , 2005 .

[9]  C. Wittwer,et al.  Real-time fluorescence genotyping of factor V Leiden during rapid-cycle PCR. , 1997, Clinical chemistry.

[10]  C. Wittwer,et al.  Rapid Cycle Real-Time PCR , 2001, Springer Berlin Heidelberg.

[11]  R. Myers,et al.  Detection and localization of single base changes by denaturing gradient gel electrophoresis. , 1987, Methods in enzymology.

[12]  M. Fixman,et al.  Theory of DNA melting curves , 1977, Biopolymers.

[13]  C. Wittwer,et al.  Fluorescein-labeled oligonucleotides for real-time pcr: using the inherent quenching of deoxyguanosine nucleotides. , 2001, Analytical biochemistry.

[14]  C. Wittwer,et al.  Continuous fluorescence monitoring of rapid cycle DNA amplification. , 1997, BioTechniques.

[15]  C. Wittwer,et al.  Rapid β-Globin Genotyping by Multiplexing Probe Melting Temperature and Color , 2000 .

[16]  Carl T. Wittwer,et al.  Rapid Cycle Real-Time PCR — Methods and Applications , 2002, Springer Berlin Heidelberg.

[17]  S. Dobrowolski,et al.  Optimization of an automated DNA purification protocol for neonatal screening. , 1999, Archives of pathology & laboratory medicine.

[18]  K. Elenitoba-Johnson,et al.  Solution-based scanning for single-base alterations using a double-stranded DNA binding dye and fluorescence-melting profiles. , 2001, The American journal of pathology.

[19]  William H. Press,et al.  Numerical Recipes in C, 2nd Edition , 1992 .

[20]  C. N. Gundry,et al.  Rapid F508del and F508C assay using fluorescent hybridization probes. , 1999, Genetic testing.

[21]  V. Burland,et al.  Use of a DNA toolbox for the characterization of mutation scanning methods. I: Construction of the toolbox and evaluation of heteroduplex analysis , 1999, Electrophoresis.

[22]  William H. Press,et al.  Numerical recipes in C , 2002 .

[23]  D. Persing,et al.  Diagnostic molecular microbiology : principles and applications , 1993 .