Identification of Hanwoo and Holstein meat using MGB probe based real-time PCR associated with single nucleotide polymorphism (SNP) in Melanocortin 1 receptor (MC1R) gene

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(1)³¤¥Èó. * .* a. (2005) 45 1 Korean J Vet Res(2005) 45(1) : 25~28. ²ÎïN.$3F*¶~4/1fNR.(#QSPCFö8.R SFBMUJNF1$3jÏRRÖG)PMTUFJOG~$ê. ;9êÁB%ÁÒ¢ *ÎL >~ ²Òß: 2005j 2ú 5¢). (. Identification of Hanwoo and Holstein meat using MGB probe based real-time PCR associated with single nucleotide polymorphism (SNP) in Melanocortin 1 receptor (MC1R) gene Sung-Do Park, Tae-Jung Kim, Jae-Il Lee* College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea (Accepted= February 5, 2005) Abstract : The melanocortin 1 receptor (MC1R) plays an important role in regulation of melanin pigment synthesis within mammalian melanocytes. Mutations within the gene encoding MC1R have been shown to explain coat color variations within several mammalian species including cattle. To develope a rapid and accurate method for the identification of Hanwoo meat, we performed a single nucleotide polymorphism (SNP) analysis in Melanocortin 1 receptor (MC1R) gene using TaqMan MGB probe-based real-time PCR. Two specific probes (one for Hanwoo and the other for Holstein and Black angus) were designed. At the 5' end of 2 TaqMan MGB probes, 6-carboxyfluorescein (FAM) was labeled for Hanwoo, and VIC for Holstein and Black angus. As a result, Hanwoo samples showed FAM-positive signal only, whereas other samples showed VIC-positive. This result suggests that the TaqMan MGB probe based real-time PCR technique would be very accurate, easy and reproducible method to discriminate between Hanwoo meat and Holstein/Black angus meat. Key words : MC1R gene, coat color, TaqMan MGB probe, real-time PCR, Hanwoo. * V. RAPD marker~ Ò*Wj BF~¶ s [6]f SCAR(sequence characterized amplified regions) marker¢ Ï~ ÖG $êj ê®b¾ ;{ \ª Ú J . 2000j êöº " ² ÎïN F*¶ Melanocortin 1 receptor(MC1R)¢ Ï 6ê» \> ® . MC1Rf ¢ò~ {Ö 5 Wj ¶ ~º ^Ò >ÏÚB 'ïö ~º phaeomelanin .ï $º Mïö ~º eumelanin~ ï²W .ö 7º j [7, 15]. ; [4]f MC1R ~ PCR-RFLP(restriction fragment length polymorphism) marker¢ Ï ÖG $ê»j ~& . ¯ PCR ÃB MC1R genej Bse118 I, Msp I, Aci I~ ^. ]ÚÖ V~ &¦ªf ÖG <²G(Holstein), Ò ¢¦ >«G ²j¶ ÖGj z F^ b ¢¦öB <²G ÖGb z7~ $ > ® . ¦; FÛj ".~V *B > j* ÖG <²G 5 >«Gj 6ê~V * VF. BB>Ú N © Ò . 7 PCRj Ï~º DNA ; ªCV»bB RAPD(random amplified polymorphic DNA)º '« ÿ>~ F*ªC 5 « $º ®« êö 9² wÏ>Ú zº [2, 14], Ö~ Ò*W¾ ;{W Ôj Ïz& ÚJ . $ . *Corresponding author: Jae-Il Lee College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Korea [Tel: +82-62-530-2854, Fax: +82-62-530-2857, E-mail: jaeil@chonnam.ac.kr]. 25.

(2) ;9êÁB%ÁÒ¢. 26. &æ B Î² ¾Ò~ ÖG" <²GöB ''. DNA band¢ {~& . O»f *~ RAPD ¾ SCAR O»ö j~ ;{~ n;' ªC Ö" ¢ áj > ®æò PCR ê DNA ;B, Ò enzyme ¾Ò";j ê agarose gelö *V'ÿj ¢ ~º ®æ ®î . $ ; [5]f MC1Rö & PCR-SSCP(single-strand conformation polymorphism) V»j Ï ÖG $ê»j ~& . O»ö B Öf <² *ö N¢ º ®« ß' SSCP F*¶;j {~& . º RFLPfº Ò B Î ²& jºì *Þ~² òæ FBB F*¶~. ;Wj ç7 ¦Â > ®b¾, polyacrylamide gel & j 5 "ï b *~ ^B 5 Ò*W ÎÚ æº 6 ®î . ö PCR-RFLP ªCO»j Ï~ ' »«öB~ Îï *; æf MC1R F* ¶;*~ &ê& , >î [1, 3, 4, 9]. SNP(single nucleotide polymorphism)º ~¾~ "V& deletion, insertion $º substitution >Ú ¾æ¾º ©b human genomeöB &Ë ôf æ~ öb [16] î÷¾ £b~ BBj * ôf & Úæ ® [11, 13]. ÖÒº j V. ê» >Ú V ; *~> ®«ê ê ÚJÚ ^ B¢ Ö~V *~ ÖG" <²G 5 Black angus Gj f *Úö ;{® ª > ®º O»j d ¶ ~& .. Òò 5 O». Òò ö ÒÏB Òòº ê»ËöB ï" ¦ fï b~ Holstein 20îÒ, ÚÎ& j*® ¦f Black angus 5îÒ, Ò j* .ï~ Ö 20îÒ, ï" .ï~ b« Hereford(>«Ö) 10îÒ¦8 "Gj j~& .

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(4) MC1R F*¶(AF445642)~ 559®öB 576®r æ¢ forward primer(5'-GCTGGAGACGGCAGTCAT-3') DNA Miller [12] phenol/chloroform DNA TE buffer(10 mM Tris-Cl, 1 mM EDTA, pH 7.4) 50 µg/ml . DNA 1% agarose gel . . ®öB ®ræ¢ ÒÏ~ ~ Öbj Ö~¶ ~&. º Öö ®Ú ® ~ Ö¶B j ~º f Ò~º j ~º ¢ Ï ~&. ö ÒÏ>º ;7 f ö & ÚÏf ö ~ J«B : ® f º ¢ Ï~ &j~&. ö ~R ~ Ã # *8'ÿ PCR >wj * >wf template DNA 11.875 µl, 40X assay mix 0.625 µl, TaqMan Universal PCR master mix(2X) 12.5 µl C 25 µl ;~& . Real-time PCR f ABI Prism 7000 sequence detection system(Applied Biosystems, USA)j Ï~ >¯>îº PCR >¯ f 95 CöB 10ª* pre-denaturation ê 92 C, 15., 60 C, 1ª*~ 2ê PCRj 40 cycles >¯~& . PCR >¯ Â ê Ö"~ ªCf SDS 7000 software (Applied Biosystems, USA)¢ Ï~ ~& .. , 617 633 reverse primer(5'TCCAGCTGCTGCACCAC-3') 75 bp PCR . Probe 594 guanine allele FAMlabeled reporter 1 TaqMan MGB probe(5'-FAMCCAGGACACCGCCT-MGB-3') guanine allele VIC-labeled reporter 2 TaqMan MGB probe(5'-VIC-CCAGGACACCGGCCT-MGB-3') . Allelic discrimination probe 5' nuclease assay Livak [10] . primer set probe set Assays-bySM Design service (Applied Biosystems, USA) 40X assay mix .. . . o. o. o. Ö . f Ò &æ~ ¢ Ï~ ² F*¶ç ~ ¢ \ª~V * þj ~& Ö Ö ò f öBò >w j & º öBò >wj ¾æêj r > ®î. º&'b º Öf îR &æ öBò >wj & ¯ Öß' º .ï Îïj ¾æÚº öò Ö ê ö ~ Wz>Ú ö ~º ;%òj B*~& .ï~ Îï jò öº ~¾~ ò Ö Ê ~ ;%òj B*j r > ®î Ö ªC ê ;%ª ~ ²8f ö ¾æ¾ ® Ö þ ö ÒÏB B~ 5 B~ ¢ Ï~º þf B> .ï~ Îï~ F*¶ Ò ¦¢ ¦Â > ®º O»ªj r > ®î . ¯ .ïj ®« Ö¾ Hereford f FAM dye ~ Wj " > ®º >, .ï~ Îï >æ. Forward primer reverse primer, 2 TaqMan MGB probe MC1R SNP . , (round) fluorescent dye FAM Holstein(diamond) Black angus(large squae) VIC dye (Fig. 1). Hereford(triangle) FAM dye . probe DNA allele Taq DNA polymerase FAM , allele DNA probe , VIC . Table 1 . , 2 primer 2 TaqMan MGB probe.

(5) ² ÎïN MC1R F*¶~ SNPf NR MGB probeö 8.R real-time PCRj ÏR RÖG HolsteinG~ $ê. 27. pf Holstein¾ Black angusº FAM dye~ Wj " > ì VIC dye~ Wòj & .. V. Fig. 1. Genotyping of Hanwoo and non-Hanwoo cattle using novel fluorescent MGB probes. After PCR, increase in VIC and FAM fluorescence representing the presence of Hanwoo and non-Hanwoo alleles, respectively, was measured. Hanwoo (round) and Hereford samples (triangle) distributed only in FAM-positive region, whereas Holstein (diamond) and Black angus samples (large square) in VIC-positive regions. No template controls were double negative (small square). Table 1. Raw data of fluorescence intensity for allelic discrimination Sample. VIC. FAM. Sample. VIC. FAM. Han*1 Han2 Han3 Han4 Han5 Han6 Han7 Han8 Han9 Han10 Han11 Han12 Han13 Han14 Han15 Han16 Han17 Han18 Han19 Han20 Hol*1 Hol2 Hol3 Hol4 Hol5 Hol6 Hol7 Hol8 Hol9 Hol10. 0.233 0.206 0.116 0.246 0.249 0.252 0.24 0.189 0.251 0.11 0.267 0.28 0.267 0.252 0.255 0.058 0.265 0.271 0.139 0.263 0.826 0.8 0.772 1.563 0.972 0.887 0.677 1.26 1.404 0.91. 1.595 1.06 2.01 1.579 2.599 1.597 3.599 2.208 1.589 2.739 1.679 1.676 2.674 1.644 1.702 3.897 2.649 1.656 1.501 2.659 0.977 0.66 0.606 1.636 0.649 0.654 0.688 0.643 1.317 1.14. Hol11 Hol12 Hol13 Hol14 Hol15 Hol16 Hol17 Hol18 Hol19 Hol20 Her*1 Her2 Her3 Her4 Her5 Her6 Her7 Her8 Her9 Her10 Ang*1 Ang2 Ang3 Ang4 Ang5 NTC*1 NTC2 NTC3 NTC4 NTC5. 0.957 1.266 1.273 0.827 1.28 1.279 1.15 0.654 0.611 1.008 0.243 0.258 0.256 0.109 0.289 0.257 0.245 0.255 0.189 0.232 1.254 1.772 1.283 1.938 1.244 0.307 0.239 0.266 0.281 0.248. 0.603 0.624 0.648 0.795 0.646 0.687 0.577 0.623 1.116 1.072 2.627 2.643 2.636 1.851 2.63 1.606 1.795 2.61 1.997 2.572 0.572 0.633 0.613 0.63 0.669 0.583 0.58 0.567 0.581 0.576. *Han: Hanwoo, Hol: Holstein, Her: Hereford, Ang: Black angus, NTC: no template control. ]ÚÖ V 7 &Ë ô FÛ> ®º ©f ÖG <²G . Gj $ röº ]ÚÖ V 7 ÖGæ <²Gæö & ;{® « © j ~Z ~ ®æò ÖÒ¾¢ ²j¶ ~ ÖG F ^ ãË r^ö <²Gb «~æ p¾ ÖGb :ú «~º ¦; FÛ W¯~ ® . ÖG <²G~ $ê»ö & jºW Ò> Ú ôf \& Úæ ® . ß® "öº ²~ ÎïF*¶ Melanocortin 1 receptor(MC1R) gene~ SNP¢ Ï $ê» \> ® . &' © BÎ²¢ Ï PCR-RFLP» [4] º Ò* W ;{Wf Ú¾æò, PCR êö BÎ²¢ ¾Ò¢ ~º ®æ ® *V'ÿj ¢~ º F * J¾ Ò, gel &j;çö F bî~ /b ~ ^B& ®î . $ PCR-SSCP »j Ï $ê» [5]f RFLPfº Ò BÎ²¾Ò ;f jºìæò *V'ÿö ÒÏ>º polyacrylamide gel¾ silver staining agarose gel¾ ethidium bromide stainingö jB B; Ç~, * J¾ Ò, ôf jÏ Ëj& jº~ . ö RAPD O »j Ï þ [2, 14]f Ö~ Ò*W¾ ;{ W Ôj Ïz& ÚJ . $ RAPD marker ~ Ò*Wj BF~¶ s [6]f SCAR(Sequence characterized amplified regions) marker¢ Ï~ Ö G $êj ê®b¾ ;{ \ª ÚJ . F þöBº TaqMan MGB probe f 2B~ primer set¢ Ï real-time PCR O»j Û ³~ ;{ \ê»j ~& . TaqMan MGB probe VFf fluorescence resonance energy transfer (FRET)ö V.¢ v ® [8]. PCR ê¯ 7 Taq DNA polymeraseº templateö binding~ ®º probef 7~ ² > 5'-3' exonuclease activityö ~ probe~ 5' ã j B~V ·~B 5'ö label>Ú ®º ;%bî (VIC $º FAM)j B . ;%bîf Wz> WzB ;%bî detectionB . O »f ' ;%bîö ~ ;%ê~ Ã&¢ Û d¶ ~º "VB~ Ò ¦¢ ªC > ®² B . Ò label>Ú ®º ;%bî~ «~& ·î> d¶ ~º "VB~ ·Wê Ã&F > ®º Ë6 ® . ²~ ®« \ê ;' ßûö ~~º © æ. . .

(6) ;9êÁB%ÁÒ¢. 28. ræ~ ¢>' O»ÚB ê»~ V ; *~> ®«ê êf ~ ®&Ë® . $ FÛ ;öB ÖG <²G ÖGb z7 $ >º ãÖ& B~& . V¢B þöB ÒÏ TaqMan MGB probe¢ Ï real-time PCR O»f 2* Ú ö B> ÖGj <²G 5 Black angusG «{® \ê > ®º ;{~ *Þ~ Ò*W ®º 6ê» ¢ 6B . ~æò þÖöB :f ? ï bÎ«(Hereford ) B>ïÖ(Ö)~ ªêf . þ O»b ®&Ë~ . ï ~ Îï D ®« B>ïj \ª~º O»f jç ^ê'b {ãB :& ìV r^ö ÖÒ¾¢~ >«GÖ ãÖ ï bÎ«(Hereford ) ®rj 6n ^ Bö & \& z º\B > ®Æ . ~æò \O»~ wÏf ·»³& 5 ²j¶¢ ^~ FÛ; 7 B~º Ö z7Gj N V Ëj WzÊV ²j¶f ·»³&~ ãB' ¶. 5 b¢ * ÖÖë~ ãçKj ;zÒ > ® b ¾j& FÛ; 7 <²G ÖGb z7> º ^B6j Oæ > ®j ©¢ 6B . . ^^ò. Bò JL ;MÖ *, J9«, ;¢;, ç 1. ,BãÎ , RÒÏ, , , . ² ®«ê Melanocortin Receptor 1(MC1R) F*¶~ F*¶; nêö 42, 735-744. "\÷S. ]ÿ>¶ö²æ RÒÏ, Z~. RAPD2000,8»j Ï 2. , 8~ ®«(ÖG, FÖG (HolsteinG), >«ÖG) \ 651-660. 37,Ï ª9. ]»Ö²æ >, ·î, ;ß1995, V, JÚ ·+, */, J JÚÏ 3. 9« B , B¢ ¢ , . Ö, B"Ò¾MÖ, MÎzÖf .Î zÖöB~ MSH Receptor(MC1R) F*¶~ F*¶ ; 5 nê jL. ]ÿ>¶ö²æ 2000, 42, 253-260. ;~, BÖ, B>, Rç8. ² ÎïN F*¶ 4. MC1R~ PCR-RFLP Marker¢ Ï ÖG $ê. ;]ÿ>¶ö²æ ~, BÖ, B>2000, Rç42, 8. 379-390. PCR-SSCP 8»j 5. , Ï ² MC1R F*¶~ ;W ªC 5 ÖG I6ê^. ]ÿ>¶ö²æ ;¢; Bò, B\2001, *, JL43,45-52. B;., ` 6. ÷× R , ÒÏ , , , . ®« ßWj Ï Ö $ê æ ¶ BB. ]ÿ>F*G«²æ 1998, 2, 107-114. 7. Cone RD, Lu D, Koppula S, Vage DI, Klungland K,. 8.. 9.. 10.. 11.. 12.. 13.. 14.. 15.. 16.. Boston B, Chen W, Orth DN, Pouton C, Kesterson RA. The Melanocortin Receptors : Agonists, Antagonists, and the Hormonal Control of Pigmentation. Recent Prog Horm Res 1996, 51, 287-317. de Kok JB, Wiegerinck ET, Giesendorf BA, Swinkels DW. Rapid Genotyping of Single Nucleotide Polymorphisms Using Novel Minor Groove Binding DNA Oligonucleotides (MGB Probes). Hum Mutat 2002, 19, 554-559. Joerg H, Fries HR, Meijerink E, Stranzinger GF. Red coat color in Holstein cattle is associated with a deletion in the MSHR gene. Mamm Genome 1996, 7, 317-318. Livak KJ. Allelic discrimination using fluorogenic probes and the 5’ nuclease assay. Genet Anal 1999, 14, 143-149. McCarthy JJ, Hilfiker R. The use of single-nucleotide polymorphism maps in pharmacogenomics. Nat Biotechnol 2000, 18, 505-508. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988, 16, 1215. Nebert DW. Pharmacogenetics and pharmacogenomics : Why is this relevant to the clinical geneticist? Clin Genet 1999, 56, 247-258. Smith EJ, Jones CP, Bartlett J, Nestor KE. Use of randomly amplified polymorphic DNA markers for the genetic analysis of relatedness and diversity in chickens and turkeys. Poultry Sci 1996, 75, 579-584. Vage DI, Klungland H, Lu D, Cone RD. Molecular and pharmacological characterization of dominant black coat color in sheep. Mamm Genome 1999, 10, 39-43. Wang D, Fan J, Siao D, Berno A, Young P, Sapolsky R, Ghandour G, Perkins N, Winchester E, Spencer J, Kruglyak L, Stein L, Jsie L, Topaloglou T, Hubbell E, Robinson E, Mittmann M, Morris M, Shen N, Kilburn D, Rioux J, Nusbaum C, Rozen S, Hudson T, Lipshutz R, Chee M, Lander E. Largescale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome. Science 1998, 280, 1077-1082..

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