Vol. 5 (1996): 541-546. Detection of bovine foetal DNA from amniotic fluid using the polymerase chain reaction Jaana Peippo and Peter Bredbacka Agricultural Research Centre ofFinland, Institute ofAnimal Production, FIN-31600 Jokioinen, Finland, e-mail: jaana.peippo@mtt.fi The aims of this study were to evaluate the amount of amniotic fluid required for diagnosis of sex, milk protein and microsatellite variants by polymerase chain reaction (PCR) and to review methods for isolating DNA from the amniotic cells. Uterine and foetal tissues were used as controls, and milk protein and microsatellite variants to check contamination of maternal cells in the PCR, The results showed that the samples do not need to be purified after DNA release from the amniotic cells and that as little as 0.5-1.5 ml of amniotic fluid is sufficient for reliable diagnosis by PCR. Key words: PCR, amniotic cells, sex determination, kappa-casein, microsatellites ntroduction The polymerase chain reaction (PCR) (Saiki et al. 1988) offers a powerful tool for foetal diag- nosis, allowing detection of small quantities of foetal cells from amniotic fluid withoutprior cell culture. In humans, foetal cells from the first trimester amniotic fluid have been widely used for various PCR-based diagnostic purposes in- cluding determination of sex (Pinckert et al. 1989, Kurauchi et al. 1992), solving the ques- tion of paternity (Nata et al. 1993), and detec- tion of pathogenic organisms such as toxoplas- ma gondii (Dupouy-Camet et al. 1990, Grover et al. 1990) or human pavrovirus 819 (Koch and Adler 1990) and for estimation of chromosome 21 copy number by quantitative PCR (von Egg- eling et al. 1993). Methods for PCR screening of a variety of infectious agents are also availa- ble in veterinary medicine (for review, see Pfef- fer et al. 1995), but have not yet been used very often at the prenatal stage. In cattle, prenatal di- agnosis by PCR has been used for detecting bo- vine citrullinaemia (Healy et al. 1993) and foe- tal sex (Kadokawa et al. 1995) and for determi- nation of sex and transgene incorporation from a foetus produced by DNA microinjection at the pronuclear stage (Hyttinen et al. 1994). In cattle, there are situations in which it would be valuable to know the sex of the foe- tuses ofpregnant heifers. This can be ascertained by examining the foetal genitals with ultrasound. However, the time within which such an exami- © Agricultural and Food Science in Finland Manuscript received June 1996 541 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=CYojOSl5K7eV9ZXn.9otCbMSYlaoFUy-9OW7ABw.GJ9RPyhnN8efcqlrBcgZ9faMcDP7jk0EBPQrIYXDN-aQtYdiveD9EMDEteNopjYKcsK1XUdnuViK-ZjAL-KyRlRAttfXyxmur8crz07Ai5Qy9mpPPS9fBV1XEtE9t-t8PMvsA2Ox0MjZ9B-dtmZWblXvO1bKHIoTIL3-q5UK_6paOi-94w9gqyU0MKOz4huNdn2YDGoQp4RIbpxhwJUYJzCsBBco8xu-kudsYrPqIp3JUIFaVCOaAmh69vWjUDbCAJaKPg6FG8iW7LUl9j7JLXk-RQQckas Peippo, J. & Bredbacka, P. Detection of DNA from amnioticfluid nation can be carried out is short, and usually several examinations have to be made to achieve efficiency approaching 100% (Curran 1992). Thus it may often be both more practical and safer to analyse cells from the amniotic fluid. Possible risks for pregnancies caused by amnio- centesis in cattle have been described by Leibo and Rail (1990). In cattle, an amniotic fluid sample can be tak- en from a living animal either through a flank incision at 2 to 5 months of pregnancy (Leibo and Rail 1990, Healyetal. 1993, Hyttinen et al. 1994) or transvaginally at 2 to 9 months of preg- nancy (Kadokawa et al. 1995) under local an- aesthetic. The advantage of using amniotic rath- er than embryonic cells is that loss ofpregnancy is avoided as there is no need to manipulate the embryo. Moreover, the timing of postimplanta- tion diagnosis is not so strictly limited as is that of preimplantation diagnosis. Amniotic fluid analysis can also be used to complement preim- plantation diagnosis, and unwanted foetuses can be detected and aborted. In this study, cells were harvested by cen- trifugation from various amounts of amniotic fluid collected from uteri of slaughtered preg- nant cows. The amniotic fluid samples were then used for diagnosis ofsex (Y-chromosomal DNA) by PCR. To distinguish between foetal and ma- ternal cells, analyses of K-casein variants (A, B and E) and, when necessary, microsatellite loci were performed. Material and methods Sample collection and preparation Ten uteri from pregnant cows were collected at a slaughterhouse. The uteri were carefully dis- sected in the laboratory and samples from each foetus and uterus and the amniotic fluid were collected. The amniotic fluid was collected through the amniotic membrane using an 18 G needle attached to a 10 ml syringe. DNA was purified from < 5mm3 pieces of foetal and uter- ine tissue by the procedure used for purifying DNA from human nucleated cells (Miller et al. 1988) From the first five foetuses, 1.5-10 ml replicates of amniotic fluid were collected and purified after proteinase K treatment (Higuchi 1989) to increase DNA yield. The purified DNA was diluted with 100 pi of distilled water. From all except the first three uteri, 0.5-1.5-ml amni- otic fluid samples were also collected and these small volumes were treated with 25-100 pi of proteinase K solution after being harvested by spinning. After proteinase K inactivation (10 min at 98°C), these latter samples were used for di- agnosis without further purification. Large vol- umes (in 10-ml tubes) were centrifuged at 3000 G and small volumes in eppendorf tubes at 13 000 G, all at room temperature for 30 min. All amni- otic fluid samples were stored frozen at -20°C before analysis. One pi of the purified and 1 or 5 pi of the unpurified amniotic fluid were used for a PCR reaction. Diagnosis by PCR All foetuses were diagnosed for phenotypic sex and by PCR. All PCR samples (uterus, foetus and amniotic fluid replicates, 1 pl/each PCR re- action) were diagnosed for sex according to Bredbacka and Peippo (1992) using the embryo sexing method based on restriction fragment length polymorphism analysis of the ZFY/ZFX locus (Fig. 1). Amplifications were performed using an MJ Minicycler (MJ Research, Inc., Watertown, MA, USA) To verify the origin of DNA (foetal or ma- ternal), the samples (1 pl/each PCR reaction) were also diagnosed for milk protein variants, K-casein A, B and E alleles (Fig. 2), according to Medrano and Aguilar-Cordova (1990) with modifications describedby Velmala et al. (1993). Amplifications were performed using the MJ Minicycler. If there was no difference between the foe- tus and the dam in either sex or K-casein vari- ants, samples were studied for microsatellite 542 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 541-546. polymorphism using three different sets ofprim- ers: BoLA (Creighton et al. 1992), and HEL 5 and HEL 10 (Kaukinen and Varvio 1993). The latter two primer pairs were amplified in the same reaction in a final volume of 25 pi. The final assay conditions were: 50 ng of sample DNA or 5 pi of centrifuged and proteinase K- treated amniotic cells, 0.8 mM dNTPs (Finnzymes, Espoo, Finland), 10 pmol of each primer and 1 IU of thermostable DNA polymer- ase (DynaZyme™, Finnzymes) in PCR buffer (10 mM Tris-HCI (pH 7.4 at 25°C), 1.5 mM MgCl2, 50 mM KCI, 0.1% Triton X-100; Finnzymes). Samples were amplified using a PTC-100™ Pro- grammable Thermal Cycler (MJ Research, Inc.) as follows: 5 min initial denaturation at 94°C followed by 30 s at 94°C, 1 min at 55°C and 35 s at 72°C for 27 cycles. A final extension at 72°C for 8 min completed each amplification session. The fluorescein- labelled PCR products were separated on 6% denaturing PAGE gel (Ready- Mix, Pharmacia, Uppsala, Sweden) using the Automated Laser Fluorescent DNA Sequencer (Pharmacia). Size was determined with an inter- nai size standard included in each lane. The gels were analysed using the Fragment Manager Vl.l program (Pharmacia). Results Five of the 10uteri collected contained a female and five a male foetus. Sexes were easily identi- fied from the phenotype of each foetus of 9 to 33 cm in size (i.e. approximately 70 to 120 days of age according to Noakes 1986). Amplifica- tion was successful on all the replicates of am- niotic fluid analysed, and correct signals were obtained each time except that a single replicate from two foetuses with original volumes of 10 ml did not result in any amplification in PCR. In these cases the pellets ofcells were probably lost during the purification process. The results for sex, K-casein and microsatellite typing are sum- marized in Table 1. Fig. I. Banding patterns from male (lanes 1-8) and female (lane 9) samples after amplification of the ZFY/ZFX loci followed by digestion of the resulting product with Pstl restriction endo- nuclease. Lines 1-7represent replicates of am- niotic fluid samples; 0.5 ml (lanes 1-3), 1 ml (lanes 3-6) and 1.5 ml (lane 7) aspirated origi- nally. Lanes 8-10 are controls: foetus, dam and a negative control (sample replaced with wa- ter), respectively. Lane 11 is a molecular size marker. (Photo: Jaana Peippo). Fig. 2. An example of kappa-casein genotypes after amplification of thekappa-casein locus fol- lowed by digestion of the resulting product with Haelll (lanes 3,5,7 and 10) and Hinfl (lanes 2, 4, 6 and 9) restriction endonucleases. Lanes 2- 3 and 4-5 represent amniotic fluid replicates, lanes 6-7 foetus and 9-10 dam, respectively. The kappa-casein genotypes are BE for the foe- tus and AE for the dam. Lane I is a negative control, where the sample DNA is replaced with water and lane 8 is a molecular size marker. (Photo: Jaana Peippo). 543 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Peippo , J. <£ Bredbacka, P. Detection ofDNA from amnioticfluid Table 1. Results of sex, kappa-casein (K-Cn) and microsatellite analysis of foetuses and their dams. Date Foetus Fluid Sex: K-Cn: Microsatellite (dam;foetus): size (cm) (ml) phenot/PCR dam/foetus BoLA HEL 5 HEL 10 26.5 13.5 4xlo F/F BE/AB n.a. n.a. n.a. 07.6 10.5 6xlo F/F AA/AB n.a. n.a. n.a. 08.6 33.0 3xlo M/M AA/AA n.a. n.a. n.a. 14.6 16.0 Ix 3 M/M AB/AB n.a. n.a. n.a. 4x1.5 26.7 18.5 2xlo F/F AA/AE n.a. n.a. n.a. 6x1.5 6xl 02.8 9.0 6x1.5 F/F AE/AE 124/134;126/134 152/162; 162/162 102/102;102/102 6xl 13.0 6x1.5 M/M AE/BE n.a. n.a. n.a. 6xl 03.8 12.5 6x1.5 M/M AA/AE n.a. n.a. n.a. 6xl 16.5 6x1.5 F/F AA/AA 126/136;126/130 152/164; 152/162 102/108;108/108 6xl 09.8 25.0 2x1.5 M/M AA/AA n.a. n.a. n.a. 4xl 6x0.5 n.a.= not analysed Discussion We show here that as little as 1 pi of proteinase K-treated amniotic fluid (0.5 ml aspirated origi- nally) contains a sufficient amount of foetal cells for analysis of single copy genes by PCR fol- lowed by restriction fragment length polymor- phism analysis. Furthermore, the amniotic fluid sample DNA does not need to be purified; on the contrary, cellular material may be lost dur- ing purification. The uniform and strong bands on the electrophoresis gels imply that parallel samples contained a cell number clearly exceed- ing the critical number for successful PCR. Hence the protocol may not be sensitive to var- iations in cell concentrations, at least in unpuri- fied samples. Maternal cell contamination is a potential source of misdiagnosis in amniotic fluid assays. Benn and Hsu (1983) reported that such contam- ination was relatively low (0% to 0.543%) in humans. We used molecular markers to verify that the DNA analysed was of foetal rather than maternal origin. In all instances, the foetal ori- gin of DNA could be confirmed by either sex determination, or K-casein or microsatelliteanal- ysis. Highly polymorphic microsatellite loci are ideal for this purpose and have previously been used in humans, too (Rebello et al. 1994,Smith et al. 1995). In our study only foetal membranes were penetrated for aspiration of amniotic fluid with a needle. In practice, however, aspiration may have to be performed transvaginally, a pro- cedure that certainly increases the risk of mater- nal cell contamination. When samples are taken for sex determination it is, however, unlikely that minor contamination will inhibit the amplifica- tion of Y-chromosomal DNA. Consequently, identificationof foetal DNA in the sample should be sufficient for successful analysis. The PCR approach can be used over a longer period during pregnancy than sexing of foetuses by ultrasound. We have amplified Y-cromosom- 544 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 541-546. al DNA from the amniotic fluid of a foetus 4 cm in size, equivalent to an age of 45-50 days, i.e. too young to be diagnosed by ultrasound. Application of the postimplantation diagno- sis described here may be useful in some com- mercial and research situations, e.g. when preg- nant heifers are to be sold or the purity of X- or Y-separated sperm populations has to be tested after artificial inseminations. The ethical aspects of amniocentesis should also be considered. If abortion may be a conse- quence of testing amniotic fluid samples, one should also consider alternative approaches, such as preimplantation diagnosis. Acknowledgements. We wish to thank the LSO abattoir in Forssa for providing the uteri and AnneliVirta forperform- ing the fragment analysis with the ALF. The technical as- sistance of Juha Kantanen, Reija Laitinen and Tuula-Mar- jatta Nieminen is greatly appreciated. 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SELOSTUS Sikiön DNA;n tunnistaminen naudan sikiövedestä polymeraasiketjureaktion avulla Jaana Peippoja Peter Bredbacka Maatalouden tutkimuskeskus Sikiövedestä tehtävä diagnostiikka on tullut nopeam- maksi ja tehokkaammaksi viime vuosikymmenellä keksityn polymeraasiketjureaktion (PCR) myötä. DNA:n voi nyt analysoida suoraan aspiroidusta sikiö- vesinäytteestä ilman soluviljelyä. Ihmislääketietees- sä PCR:ään perustuva sikiövesidiagnostiikka on ol- lut käytössä jo useiden vuosien ajan. Valmius myös naudan perinnöllisten sairauksien ja taudinaiheutta- jien toteamiseksi on jo olemassa, mutta menetelmiä ei ole vielä otettu yleisesti tähän käyttöön. Tässä ko- keessa haluttiin selvittää, kuinka pienestä sikiövesi- näytteestä sukupuolenmääritys voidaan luotettavasti tehdä, ja miten näyte on puhdistettava analyysiä var- ten. Tutkimusta varten teurastamolta haettiin 10 koh- tua 70-120 päivän ikäisine sikiöineen. Laboratoriossa kerättiin näytteet sikiövedestä, sikiöstä ja kohdusta. Sikiövedestä määritettiin sukupuoli ja kappa-kaseii- ni -tyyppi, jotta näytteen voitiin osoittaa sisältävän sikiön soluja. Jos emän ja sikiön välillä ei ollut kum- massakaan em. analyysissä eroa, tehtiin vielä mikro- satelliitteihin eli DNA:n toistojaksoihin perustuva analyysi. Mikrosatelliitteja esiintyy läpi koko geno- min ja kunkin mikrosatelliittilokuksen muuntelu eri yksilöiden välillä on suurta, joten niiden joukosta on mahdollista valita sellaiset, joiden “sormenjäljet” ovat yksilölliset. Kaikista sikiövesinäytteistä saatiin sama signaa- li kuin itse sikiöstä ja nämä molemmat poikkesivat emän signaalista. Kahdesta puhdistetusta sikiövesi- näytteestä katosi soluaines puhdistuksen aikana, jo- ten on yksinkertaisempaa ja turvallisempaa jättää näyte puhdistamatta, koska analyysin onnistuminen ei siitä vaarannu. Tutkimuksen perusteella voidaan todeta, että ke- hittyvän sikiön sukupuoli ja tarvittaessa muitakin DNA-tasolla näkyviä ominaisuuksia voidaan luotet- tavasti määrittää suoraan 0,5-1,5 ml:stä sikiövettä il- man edeltävää soluviljelyä tai DNA:n puhdistusta. 546 AGRICULTURAL AND FOOD SCIENCE IN FINLAND