Detection of amplified Y chromosome-specific sequence by capillary electrophoresis with laser-induced fluorescence*

Transcription

1 FERTILITY AND STERILITY 1995 Americn Society for Reproductive Medicine Vol. 64, No., August 1995 Printed on cid free pper in U. S. A. Detection of mplified Y chromosome-specific sequence by cpillry electrophoresis with lser-induced fluorescence* MingSun Liu, Ph.D. t:j: Sushm Rmpl, B.S. t Rmon A. Evngelist, Ph.D.t Gregory C. Y. Lee, Ph.D. Fu-Ti A. Chen, Ph.D.t Advnced Technology Center, Beckmn Instruments Inc., Fullerton, Cliforni, nd Deprtment of Obstetrics nd Gynecology, University of British Columbi, Vncouver, British Columbi, Cnd Objective: To develop sensitive method for genetic dignosis using cpillry electrophoresis with lser-induced fluorescence. Design: Using mle DNA diluted with femle DNA s n exmple, ZFY gene from Y chromosome ws mplified specificlly by polymerse chin rection (PCR) with fluorescence-lbeled primers nd detected by cpillry electrophoresis with lser-induced fluorescence. Setting: Lser operting lbortory. Prticipnts: Humn mle nd femle DNA were extrcted from helthy humn mle nd femle subjects. Interventions: None. Min Outcome Mesures: The concentrtion of humn DNA ws determined by using spectrophotometer t n bsorbnce of 6 nm. Result: Deoxyribonucleic cid frgments mplified from single copy of ZFY gene were detected by cpillry electrophoresis with lser-induced fluorescence fter 5 cycles of PCR mplifiction. Conclusion: This method is potentilly pplicble for rpid nd sensitive detection of fetl Y chromosome DNA sequence in mternl circultion nd of single-cell DNA dignosis. Fertil Steril1995;64: Key Words: Cpillry electrophoresis, polymerse chin rection, single copy DNA detection During erly pregnncy, few fetl cells hve been found relesed in mternl circultion (1). Bsed on the polymerse chin rection (PCR), the Y chromosoml DNA sequence of mle fetl cells could be mplified nd detected from mternl blood (). The mplified DNA frgments generlly were checked by trditionl gel electrophoresis, which required 1 ng DNA for detection by ethidium bromide stining, nd the procedure ws time consuming. Furthermore, to observe cler DNA signl in conventionl gel, two pirs of primer nd totl of 5 Received October 1, 1994; revised nd ccepted Mrch 9, * Supported by Advnced Technology Center, Beckmn Instruments Inc., Fullerton, Cliforni. t Advnced Technology Center, Beckmn Instruments Inc. t Reprint requests: Ming-Sun Liu, Ph.D., Advnced Technology Center, D--A, Beckmn Instruments Inc. 5 Hrbor Boulevrd, Fullerton, Cliforni 964 (FAX: ). Deprtment of Obstetrics nd Gynecology, University of British Columbi. Vol. 64, No., August 1995 to 7 mplifiction cycles hve been designed for the PCR process. However, ech pregnnt womn hs vrying mount of fetl cells in circultion t the erly pregnncy stge tht might cuse either flse-negtive result becuse of the low sensitivity detection or flse-positive result by nonspecific mplifiction becuse of the high number ofpcr cycles. To chieve greter performnce, cpillry electrophoresis equipped with lser-induced fluorescence hs been introduced s tool for rpid nd sensitive DNA nlysis (). In this report, we used the cpillry electrophoresis-lser-induced fluorescence system to detect PCR-mplified DNA from single copy of ZFY gene. This pproch could be used s tool for fetl DNA detection in mternl circultion nd genetic dignosis from single cell. MATERIALS AND METHODS To exmine the single copy detection of ZFY gene, mle DNA ws diluted serilly with femle DNA for Liu et I. Techniques nd instrumenttion 447

2 M._±& Tble 1 Dilution Rtio of Mle DNA to Femle DNA in Ech Smple Smple no DNA rtio mle:femle 1: (positive control) 1:1 1:1 1:1, 1:1, 1:1, 1:, 1:4, 1:, 1:1,6, 1:,, :1 (negtive control) the PCR mplifiction (Tble 1). Two oligonucleotide primers used for mplifiction of humn Y chromosome-specific ZFY gene sequences were 5'CTCTCA GTTCACACAAAGG' nd 5' GCTTGTAGACACA CTGTTAGG' nd prepred by OLIGO 1 DNA synthesizer (Beckmn, Fullerton, CA). An N-TFA C6-AminoModifier (Clontech, Plo Alto, CA) ws ttched to the 5' end of oligonucleotides during the synthesis nd conjugted with Cy5 dye (Biologicl Detection Systems, Inc., Pittsburgh, PA) (4). Cy5 (Amx = 65 nm, = 15, M- I cm- I ) is cynine dye tht is useful s fluorescent lbel for biologic compounds. The detection sensitivity for Cy5 in the cpillry electrophoresis-lser-induced fluorescence system is 1-11 M. Deoxyribonucleic cid mplifiction by PCR ws performed ccording to the protocols described previously (4). Briefly, the 1 ml of mplifiction rection mixture contins 1 mg humn mle genomic DNA, pmol ech of the two Cy5-lbeled primers,.5 mm ech of four deoxyribonucleoside triphosphtes, nd. U of Tq DNA polymerse (Boehringer Mnnheim, Indinpolis, IN). The rection ws crried out in.-ml microcentrifuge tubes nd overlid with minerl oil. The smple underwent n initil denturtion t 94 C for 5 minutes, followed 1 - A Primers - 6-4, per mplified ZFY DNA r Figure 1 (A), Electropherogrm of PeR-mplified ZFY DNA frgments of smple 1 by cpillry electrophoresislser-induced fluorescence. The mplified DNA frgments (7 bp) nd unused primers re indicted by rrows. (B), Electropherogrm of mixtures of nd 1 bp DNA ldders. Moleculr sizes indicted on the top of ech pek re 1, bp;, 1 bp;, bp; 4, bp; nd 5, 4 bp, respectively. 44 Liu et l. Techniques nd instrumenttion Fertility nd Sterility

3 I -"'" ' ' '".. - I 1 - ' >. - >- ::::... (I'J 'en C. C Q) CD C.S.4.5 Y Q).1 CD. C,.) J\...-. U C C Q) CD C,.) -.1 U (I'J en Q) CD. 4 y :J 1 ::l.1 ;:;:: ;:;::. CD Q). > > :::; :::;. ell Q5 Q) :::: ::: y Figure Detection ofpcr-mplified ZFY DNA frgments from smples 1 to by cpillry electrophoresis-lser-induced fluorescence. The mplified ZFY DNA frgments re indicted by rrows. 1 to correspond to smples 1 to mentioned in Tble 1, respectively. Vol. 64, No., August 1995 Liu et I. Techniques nd instrumenttion 449

4 by 5 cycles of templte denturtion t 94 C for 1 minute, primer-templte nneling t 55 C for 1 minute, nd primer extension t 7 C for 1 minute. At the end of the PCR cycles, the 1-minute incubtion t 7 C ws dded. Amplified DNA smple (7 bse pirs [bps] in length) ws nlyzed by cpillry electrophoresis-lser-induced fluorescence or by grose gel electrophoresis (.% grose contining.5 mglml ethidium bromide). For the DNA nlysis by cpillry electrophoresislser-induced fluorescence, the PlACE system 1 cpillry electrophoresis equipped with LIF detector (Beckmn, Fullerton, CA) ws used s stndrd equipment. The cthode ws set on the injection side nd the node ws set on the detection side. A 75 mm ID X 47 cm totl length (4 cm to detector) coted cpillry column filled with Beckmn dsdna 1 gel buffer (Beckmn) ws used to seprte dsdnafrgments t C. A 1 mwred HeINe lser source (Melles Griot, Irvine, CA) combined with notch filter nd bnd-pssed filter provided excittion t 6 nm nd emission t 667 nm to detect Cy5-lbeled mplified DNA frgments, which were injected by pressure into the cpillry for 99 seconds fter the PCR process. The seprtion ws run t field of 65 V/cm nd the electropherogrms were recorded by PlACE. softwre. Deoxyribonucleic cid nd 1 bp ldders (GenSur, Del Mr, CA) were used s moleculr size stndrds. RESULTS Tble 1 shows the DNA smples for PCR mplifiction nd the dilution rtio of mle DNA to femle DNA in ech smple. Smple 1 contins 1 mg of undiluted mle DNA used s positive control nd smple contined 1 mg femle DNA used s negtive control. Figure 1 shows the electropherogrm of PCR-mplified ZFY DNA sequence from smple 1 tht ws nlyzed by cpillry electrophoresis-lser-induced fluorescence. Within the 1-minute electrophoresis running period, single shrp pek of mplified Cy5-lbeled DNA ws detected clerly t the position of predicted moleculr size (7 bp). Using the electrophoretic migrtion time of smple 1 s stndrd, PCR-mplified DNA frgments from smples to were exmined by cpillry electrophoresis-lser-induced fluorescence, respectively. Figure shows the electropherogrms from smples 1 to. The mplified ZFY DNA frgments hve been detected clerly from smples to 1 (Fig., to, 1). In smple 1, mle DNA ws diluted with femle DNA t the rtio of 1:1,6,, which represents pproximtely. to 1 copy ofzfy gene in the solution. It is pprent tht single copy of ZFY gene ws detectble by cpillry electropho45 Liu et l. Techniques nd instrumenttion M Figure Agrose gel electrophoresis of PCR-mplified ZFY DNA frgments. Lnes 1 to correspond to the exmintion of 1 ml PCR-mplified DNA solution from smples 1 to listed in Tble 1. Lne M is n 1 bp DNA ldder nd the moleculr sizes (bp) re indicted on the left of the figure. resis-iser-induced fluorescence fter 5 PCR mplifiction cycles. In comprison to trditionl grose gel electrophoresis stined by ethidium bromide, the DNA signl hrdly ws detectble by ultrviolet trnsmission fter 1 to 1 dilutions (Fig. ). Bsed on our dt, the detection limit of mplified DNA in cpillry electrophoresis-lser-induced fluorescence system is estimted to be four to five orders of mgnitude lower thn tht in grose gel electrophoresis. The high sensitivity of cpillry electrophoresis-lser-induced fluorescence llows detection of single copy gene fter 5 PCR mplifiction cycles nd thus should lower the incidence of flse-positive results due to errors rising from high PCR cycle numbers. This ppliction is extremely useful for PCR mplifiction-bsed single-cell dignosis. Moreover, it is worth mentioning tht the pressure injection in cpillry electrophoresis only consumes few nnoliters of smple solution; the mplified DNA frgments from rpid nd smll volume PCR work could be nlyzed directly by cpillry electrophoresis-iserinduced fluorescence. DISCUSSION In our demonstrtion, the cpillry electrophoresis-lser-induced fluorescence system provides simple, rpid, nd sensitive method to nlyze PCRmplified DNA frgments. From our studies, one coted cpillry column my lst for month in cpillry electrophoresis-lser-induced fluorescence system nd be used for hundreds of runs. The dvntges of using cpillry electrophoresis-lser-induced fluorescence for PCR-bsed prentl dignosis Fertility nd Sterility

5 re s follows: [1] single-copy DNA sequence could be detected by using one primer set nd 5 per mplifiction cycles; [] cpillry electrophoresis-lser-induced fluorescence provides rpid detection nd gret resolution; [] the dt could be entered directly into the computer for further nlysis. In conclusion, the cpillry electrophoresis-lser-induced fluorescence system provides gret potentil for the future of DNA nlysis, especilly for DNA mplifiction-bsed genetic dignosis. Acknowledgement. Ming-Sun Liu, Ph.D., Sushm Rmpl, B.S., Rmon A. Evngelist, Ph.D., nd Fu-Ti A. Chen, Ph.D., re ll employees of Beckmn Instruments Inc. nd ll hve stock ownership in the compny through the employees' stock purchse pln. REFERENCES 1. Schroder J, de l Chpelle A. Fetl lymphocytes in the mternl blood. Blood 197;9: Lo YMD, Winscot JS, Gillmer MDG, Ptel P, Smpierto M, Fleming KA. Prentl sex determintion by PCR mplifiction from mternl peripherl blood. Lncet 199;: Lu W, Hn D-S, Yun J, Andrieu J-M. Multi-trget PCR nlysis by cpillry electrophoresis nd lser-induced fluorescence. Nture 1994;6: Chen FTA, Tusk A, Pentoney S Jr, Konrd K, Lew C, Koh E, et l. Semiconductor lser-induced fluorescence detection in cpillry electrophoresis using cynine dye. J Chromtogr 199;65:55-6. Vol. 64, No., August 1995 Liu et l. Techniques nd instrumenttion 451