High resolution DNA melting analysis: an application for prenatal control of alpha-thalassemia.
OBJECTIVE
To report the use of real-time gap-PCR using SYTO9 with high-resolution melting analysis (HRMA) in prenatal diagnosis of alpha-thalassemia 1.


METHODS
Real-time gap-PCR using SYTO9 with HRMA was performed in 33 DNA samples from chorionic villi sampling (8 normal, 16 heterozygous, and 9 homozygous) to determine the alpha-thalassemia 1 gene [normal and Southeast Asia (-SEA) allele].


RESULTS
The dissociation curve analysis in normal and - SEA allele gave a peak of T(m) at 91.80 +/- 0.14 degrees C and 88.67 +/- 0.08 degrees C, respectively. Normal genotype and homozygous alpha-thalassemia 1 showed a single peak of T(m) that corresponded to their alleles. The heterozygotes gave both peaks with higher normal peak and smaller - SEA peak. Thirty one samples showed consistent results with the conventional gap-PCR. Two samples with ambiguous results were confirmed to be maternal DNA contamination on real-time quantitative PCR and microsatellite assay. HRMA from both samples showed similar pattern to that of heterozygotes. However, they showed much smaller normal peak compared with the - SEA peak, which is in contrast to those of heterozygotes and can readily be distinguished.


CONCLUSIONS
HRMA with SYTO9 is feasible for prenatal diagnosis of alpha-thalassemia. It had potential advantage of prompt detection maternal DNA contamination.
Department of Obstetrics and Gynecology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. ssiricho@mail.med.cmu.ac.th
Supatra
S
Sirichotiyakul
Chanane
C
Wanapirak
Rattika
R
Saetung
Torpong
T
Sanguansermsri
D015193:Chorionic Villi Sampling; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics / Q000517:prevention & control
D015342:DNA Probes; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C103389:SYTO 9; D004247:DNA