Amplicon melting analysis with labeled primers: a closed-tube method for differentiating homozygotes and heterozygotes.
BACKGROUND
Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR.


METHODS
We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (T(m)). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required approximately 1 min and no sample processing was needed after PCR.


RESULTS
Polymorphisms in the HTR2A (T102C), beta-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 degrees C/s) of denatured products, followed by rapid heating during melting analysis (0.2-0.4 degrees C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in T(m) by <0.2 degrees C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5' tail of identical sequence was added to one of the two unlabeled primers.


CONCLUSIONS
High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.
Gundry
Cameron N
CN
Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132, USA.
Vandersteen
Joshua G
JG
Reed
Gudrun H
GH
Pryor
Robert J
RJ
Chen
Jian
J
Wittwer
Carl T
CT
D001483:Base Sequence; D052160:Benzothiazoles; D001798:Blood Proteins / Q000235:genetics; D003550:Cystic Fibrosis / Q000235:genetics; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics*; D003959:Diamines; D005456:Fluorescent Dyes; D005838:Genotype; D005914:Globins / Q000235:genetics; D020180:Heteroduplex Analysis; D006579:Heterozygote; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006720:Homozygote; D006801:Humans; D024101:Mitochondrial Proteins; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D011804:Quinolines; D012697:Serine Endopeptidases / Q000235:genetics; D013696:Temperature
D052160:Benzothiazoles; D001798:Blood Proteins; C505032:CFTR protein, human; D003959:Diamines; D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C098022:SYBR Green I; D005914:Globins; D004247:DNA; D012697:Serine Endopeptidases; D000074923:High-Temperature Requirement A Serine Peptidase 2