Naslov
Our Experience in Non-Invasive Prenatal Paternity Testing

Autori
Škaro, Vedrana ; Projić, Petar ; Lauc, Gordan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Biomedical Papers / Ulrichová, Jitka - Olomouc : Palacký University Press, 2012, S108-S108

Skup
2nd Central-Eastern European Symposium on Free Nucleic Acids in Non-Invasive Prenatal Diagnosis

Mjesto i datum
Olomouc, Češka Republika, 25.10.2012. - 26.10.2012

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
non-invasive; prenatal testing; paternity; Y-STR; PowerPlex Y23

Sažetak
AIMS: In order to minimize sample re-processing, which was done for most samples to amplify all of the 16 tested Y-STR loci, we made changes to our routine protocol for prenatal paternity testing. We found Centricon® and Microcon® centrifugal filter devices useful for efficient concentration of cell-free DNA (cfDNA). Also, we tested the newest Y-STR system which provides 7 additional STR loci. METHODS: Peripheral blood (9 mL) from pregnant women bearing male fetuses (n=20, gestation age was between 12 and 19 weeks) was collected into tubes containing ehylenediaminetetraacetic acid as anticoagulant. Buccal swab samples were taken from alleged fathers. Because Y-STR analysis cannot distinguish between close relatives, alleged fathers were asked to sign written statements verifying that they were not related to another alleged father. Plasma was isolated from each sample via double centrifugation protocol. DNA was extracted from plasma using QIAamp DNA Blood Maxi Kit. Extracted DNA was concentrated by ultrafiltration on Centricon® YM-100 and Microcon® YM-100 centrifugal filter devices (Millipore). Y chromosome STR loci were amplified using the AmpFlSTR® Yfiler® PCR Amplification Kit (Applied Biosystems) and PowerPlex® Y23 System (Promega). Amplified fragments were separated by capillary electrophoresis on 3130 Genetic Analyzer (Applied Biosystems) and analyzed using GeneMapperÒID-X (Applied Biosystems) software. RESULTS: Generally, more loci were amplified when samples were processed on Centricon® and Microcon® than on Centricon®alone. Similar results were noted when samples were concentrated on Microcon® compared to concentrations on the vacuum concentrator. Some samples with non loci amplified on Yfiler showed several peaks on PowerPlex® Y23 (PP Y23). Samples with partial results on Yfiler were more successfully amplified on PP Y23- yielding one or more loci more compared to Yfiler. However, since PPY23 tests 7 additional loci compared to Yfiler, overall results for this kit were even more informative (i.e., samples with 12/16 loci on Yfiler resulted with 22/23 on PPY23). With slight modifications to our routine DNA extraction method and new Y-STR kit, paternity testing was successfully performed for all 20 cases. CONCLUSIONS: Centricon® and Microcon® centrifugal filter devices efficiently concentrate cfDNA which is quite important for successful prenatal paternity testing. Although amount of fetal cfDNA present is a small fraction of the maternal cfDNA, fetal cfDNA can be successfully amplified by Y-STR kits since primers are Y-chromosome specific and therefore do not bind maternal DNA. However, by default, this type of analysis cannot be performed

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA