Views: 1,590 | Downloads:
225
| Responses: 0
| XML | Respond to this article | Alert & updates | Request permissions | Email to a friend |
Original ArticleOpen Access
Semi-Nested Taqman Real-Time Quantitative PCR for Noninvasive Prenatal Diagnosis of Bart’s Hydrops Fetalis
Pornprasert S 
,
Sukunthamala K ,
Kunyanone N ,
Sittiprasert S ,
Thungkham K ,
Junorse S ,
Pongsawatkul K ,
Pattanaporn W ,
Jitwong C
,
Sukunthamala K ,
Kunyanone N ,
Sittiprasert S ,
Thungkham K ,
Junorse S ,
Pongsawatkul K ,
Pattanaporn W ,
Jitwong C Background: Non-invasive prenatal diagnosis based on detection of fetal cell-free DNA is limited when mother and father are
both carriers for the same autosomal recessive mutation.
Objective: Develop the semi-nested Taqman real-time PCR for quantification of α-thalassemia-1 SEA type deletion allele in
plasma of α-thalassemia-1 SEA carriage pregnancies.
Material and Method: Plasma DNA was extracted from six women who carried fetuses with normal, 11 with heterozygote
α-thalassemia-1 SEA type deletion and seven with Bart’s hydrops fetalis. DNA was amplified using conventional PCR with the
primary specific primer set for α-thalassemia-1 SEA type deletion. PCR product was then subjected to the semi-nested realtime
PCR using the secondary specific primer and Taqman probe set for α-thalassemia-1 SEA type deletion. The standard
curve was constructed using ten-fold serial dilutions of conventional PCR product of the heterozygote α-thalassemia-1 SEA
type deletion.
Results: Women who carried fetuses with Bart’s hydrops fetalis displayed a trend toward higher mean copy number of
α-thalassemia-1 SEA type deletion allele vs. women who carried fetuses with normal and heterozygote, albeit not reaching
statistical significance.
Conclusion: The maternally inherited fetal allele present in maternal plasma is difficult to discern the fetal cell-free DNA from
a higher background DNA of the mother. Thus, further investigation is needed to improve the diagnosis of Bart’s hydrops
fetalis using this technique.
Keywords: α-thalassemia-1 SEA type deletion, Fetal cell-free DNA, Prenatal diagnosis, Semi-nested real-time PCR, Taqman
probe
both carriers for the same autosomal recessive mutation.
Objective: Develop the semi-nested Taqman real-time PCR for quantification of α-thalassemia-1 SEA type deletion allele in
plasma of α-thalassemia-1 SEA carriage pregnancies.
Material and Method: Plasma DNA was extracted from six women who carried fetuses with normal, 11 with heterozygote
α-thalassemia-1 SEA type deletion and seven with Bart’s hydrops fetalis. DNA was amplified using conventional PCR with the
primary specific primer set for α-thalassemia-1 SEA type deletion. PCR product was then subjected to the semi-nested realtime
PCR using the secondary specific primer and Taqman probe set for α-thalassemia-1 SEA type deletion. The standard
curve was constructed using ten-fold serial dilutions of conventional PCR product of the heterozygote α-thalassemia-1 SEA
type deletion.
Results: Women who carried fetuses with Bart’s hydrops fetalis displayed a trend toward higher mean copy number of
α-thalassemia-1 SEA type deletion allele vs. women who carried fetuses with normal and heterozygote, albeit not reaching
statistical significance.
Conclusion: The maternally inherited fetal allele present in maternal plasma is difficult to discern the fetal cell-free DNA from
a higher background DNA of the mother. Thus, further investigation is needed to improve the diagnosis of Bart’s hydrops
fetalis using this technique.
Keywords: α-thalassemia-1 SEA type deletion, Fetal cell-free DNA, Prenatal diagnosis, Semi-nested real-time PCR, Taqman
probe
Download:
PDF