Tabel Pengamatan dan Harapan Genotipe - PADA KAMBING PE, SAANEN DAN PESA DENGAN METODE PCR-SSCP

PADA KAMBING PE, SAANEN DAN PESA DENGAN METODE PCR-SSCP

Lampiran 3. Tabel Pengamatan dan Harapan Genotipe

Bangsa Populasi Genotipe

DD DE EE GH PE Ciapus (20) 0 20 0 0 Cariu (28) 0 20 0 7 Elang 45 (50) 0 20 30 0 Saanen Cijeruk (20) 0 0 0 20 Cariu (31) 0 0 0 0 Taurus DF (40) 1 37 2 0 PESA Cijeruk (7) 0 0 0 7 Cariu (25) 0 25 0 0 Balitnak (19) 4 15 0 0 Total 240 5 137 32 34 Harapan

Bangsa Populasi Nilai Harapan Genotipe

DD EE DE GH PE Ciapus (20) 5,000 5,000 10,000 0,000 Cariu (28) 3,704 3,704 7,407 0,907 Elang 45 (50) 2,000 32,000 16,000 0,000 Saanen Cijeruk (20) 0,000 0,000 0,000 10,000 Cariu (31) 0,000 0,000 0,000 0,000 Taurus DF (40) 9,506 10,506 19,988 0,000 PESA Cijeruk (7) 0,000 0,000 0,000 3,500 Cariu (25) 6,250 6,250 12,500 0,000 Balitnak (19) 6,961 2,961 9,079 0,000

37 Hardy-Weinberg Bangsa Populasi X² PE Ciapus (20) 20 Cariu (28) 69,72222 Elang 45 (50) 3,125 Saanen Cijeruk (20) 10 Taurus DF (40) 28,97873 PESA Cijeruk (7) 3,5 Cariu (25) 25 Balitnak (19) 8,081285

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Lampiran 1. Sekuens Gen GH pada Ternak Kambing (Capra hircus)

LOCUS GOTGHRA 2544 bp DNA hilinear MAM 12-JUL- 2007

DEFINITION Capra hircus gene for growth hormone, complete cds. ACCESSION D00476

VERSION D00476.1 GI:217664 KEYWORDS .

SOURCE Capra hircus (goat) ORGANISM Capra hircus

Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;

Mammalia; Eutheria; Laurasiatheria; Cetartiodactyla; Ruminantia; Pecora; Bovidae; Caprinae; Capra.

REFERENCE 1 (bases 1 to 2544)

AUTHORS Kioka,N., Manabe,E., Abe,M., Hashi,H., Yato,M., Okuno,M., Yamano,Y., Sakai,H., Komano,T., Utsumi,K. and Iritani,A. TITLE Cloning and sequencing of goat growth hormone gene JOURNAL Agric. Biol. Chem. 53, 1583-1587 (1989)

COMMENT In <Agric. Biol. Chem. 53, 1583-1587 (1989)>, they discuss that the

differences in the nucleotide sequences between the goat growth hormone cDNA and the chromosomal gene may be due to polymorphic differences in individual goats.

FEATURES Location/Qualifiers source 1..2544 /organism="Capra hircus" /mol_type="genomic DNA" /sub_species="Tokara" /db_xref="taxon:9925" /tissue_type="spleen"

/note="375 bp upstream of BamHI site.;

a genomic library using bovine growth hormone cDNA as a probe"

TATA_signal 344..349

/note="TATA box" prim_transcript 374..2169

/note="GH mRNA and introns" CDS join(432..444,692..852,1080..1196,1426..1587,1864..2064) /note="GH" /codon_start=1 /product="growth hormone" /protein_id="BAA00368.1" /db_xref="GI:217665" /translation="MMAAGPRTSLLLAFTLLCLPWTQVVGAFPAMSLSGLFANAVLRA QHLHQLAADTFKEFERTYIPEGQRYSIQNTQVAFCFSETIPAPTGKNEAQQKSDLELL RISLLLIQSWLGPLQFLSRVFTNSLVFGTSDRVYEKLKDLEEGILALMRELEDVTPRA G QILKQTYDKFDTNMRSDDALLKNYGLLSCFRKDLHKTETYLRVMKCRRFGEASCAF" exon <432..444 /number=1 intron 445..691 /number=1 protein_bind 475..480

/note="glucocorticoid receptor binding site" /bound_moiety="human glucocorticoid receptor" exon 692..852

/number=2 intron 853..1079 /number=2

34 exon 1080..1196

/number=3 variation 1148

/note="c in genomic clone; t in cDNA clone" /replace="t" intron 1197..1425 /number=3 exon 1426..1587 /number=4 intron 1588..1863 /number=4 exon 1864..>2064 /number=5 variation 2055

/note="c in genomic clone; t in cDNA clone" /replace="t"

ORIGIN

1 gggattttct gacccaggga ttaaacctga gtctcctgca tttgcagctc gattctttat 61 ggctgagcca cctgggaagc ccattcgttt ctgctacctc ccccttaaaa agaaaaccta 121 tggggtgggc tctcaagctg agaccctgtg tgtacagccc tcaggctggt ggcagtggag 181 aggggatgat gatgagcctg ggggacatga ccccagagaa ggaacgggaa caggatgagt 241 gagaggaggt tctaaattat ccattagcac aggctgccag tggtccttgc ataaatgtat 301 agagcacaca ggtgggggga aagggagaga gaagaagcca gggtataaaa agggcccagc 361 agagaccaat tccaggatcc caggacccag ttcaccagac gactcagggt cctgctgaca 421 gctcaccaac tatgatggct gcaggtaagc tcacaaaaat cccctccatt agcgtgtcct 481 aagggggtga tgcgggagaa ctgccgatgg atgtgtccac agctttgggt tttagggctt 541 ctgaatgcga acataggtat ctgcacccag acatttggcc aagtttgaaa tgttctcagt 601 ccctggaggg aagggcaggc gggggctggc aggagatcag gcatccagct ctctgggccc 661 ctccgtcgcg gccctcctgg tctctcccta gggccccgga cgtccctgct cctggctttc 721 accctgctct gcctgccctg gactcaggtg gtgggcgcct tcccagccat gtccttgtcc 781 ggcctgtttg ccaacgctgt gctccgggct cagcacctgc atcaactggc tgctgacacc 841 ttcaaagagt ttgtaagctc cccagagatg tgtcctagag gtggggaggc aggaaggggt 901 gaatccgcac cccctccaca caatgggagg gaactgagga cctcagtggt attttatcca 961 agtaaggatg tggtcagggg agtagaaatg ggggtgtgtg gggtggggag ggttccgaat 1021 aaggcagtga ggggaaccac acaccagctt agacccgggt gggtgtgttc tccccccagg 1081 agcgcaccta catcccggag ggacagagat actccatcca gaacacccag gttgccttct 1141 gcttctccga aaccatcccg gcccccacgg gcaagaatga ggcccagcag aaatcagtga 1201 gtggccacct aggaccgagg agcaggggac ctccttcatc ttaagtaggc tgccccagct 1261 ctctgcaccg ggcctggggt ggcgttctcc ctgaggtggc agagggtgtt ggatggcagt 1321 ggaggatgat ggttggtggt ggtggcagga ggtcctcggg cagaggccga ccttgcaggg 1381 ctgccccgag cccggggcac ccaccaacca cccatctgcc agcaggactt ggagctgctt 1441 cgcatctcac tgctccttat ccagtcgtgg cttgggcccc tgcagttcct cagcagagtc 1501 ttcaccaaca gcctggtgtt tggcacctcg gaccgtgtct atgagaagct gaaggacctg 1561 gaggaaggca tcctggcgct gatgcgggtg aggatggcgt tgttgggtcc cttccatgct 1621 gggggccatg cccaccctct cctggcttag ccaggagaac acacgtgggc tgggggagag 1681 agatccctgc tctctctctc tctttctagc agcccagtct tgacccagga gaaacctctt 1741 cccgttttga aacctccttc ctcgcccttc tccaagccta taggggaggg tggaaaatgg 1801 agcgggcagg agggagccgc tcctgagggc cttcggcctc tctgtctctc cctcccttgg 1861 caggagctgg aagatgttac cccccgggct gggcagatcc tcaagcagac ctatgacaaa 1921 tttgacacaa acatgcggag tgacgacgcg ctgctgaaga actacggtct gctctcctgc 1981 ttccggaagg acctgcacaa gacggagacg tacctgaggg tcatgaagtg tcgccgcttc 2041 ggggaggcga gctgcgcgtt ctagttgcca gccatctgtt gttacccctc cccgtgcctt 2101 cctagaccct ggaaggtgcc actccagtgc ccactgtcct ttcctaataa agcgaggaaa 2161 ttgcatcaca ttgtctgagt aggtgtcatt ctattctagg gggtggggtc aggcaggata 2221 gcgagaggga ggattgggaa gacaatagca gggatgctgt gggctctatg ggtacccagg 2281 tgctgaataa ttgacccggt tcttcctggg ccagaaggaa gcaggcacat ccccttctct 2341 gtgacacacc cggtcctcgc ccctggtcct tagttccagc cccactcata ggacactcat 2401 agctcaggag ggctctgcct tcagtcccac ccgctaaagt gcttggagcg gtttctcctt 2461 ccctcatcag cccaccaaac caaacctagc ctccaagagt gggaagaaat taaagcaaga 2521 caggctatga agtacagagg gaga

Lampiran 2. Protokol Ekstraksi DNA Metode Fenol (Sambrook et al., 1989)

1. 200 µl sampel darah dalam EtOH dipindahkan ke tabung 1,5 ml *50 µl sampel buffy coat

*25 mg sampel otot, digerus/dipotong kecil sampai halus 2. Tambahkan 1000 µl DW/TE

3. Kocok kuat/vorteks dan diamkan ± 5 menit

4. Sentrifuge pada kecepatan 8000 rpm selama 5 menit, buang bagian supernatan 5. Ulangi tahap 2-4

6. Tambahkan 40 µl SDS 10%

10 µl ProtK 5 mg/ml dan 1 x STE sampai 400 µl

7. Kocok pelan dalam inkubator pada suhu 55 oC selama 2 jam/homogenkan 8. Tambahkan 400 µl larutan phenol

400 µl CIAA (Chloroform; Isoamyl alkohol = 24:1) dan 40 µl 5 M NaCl

9. Kocok pelan pada suhu ruang selama 1 jam

10. Sentrifuge pada kecepatan 12000 rpm selama 5 menit

11. Pindahkan bagian DNA (bening) dengan pipet ke tabung 1,5 ml yang baru dan sisanya dibuang (± 400 µl)

12. Tambahkan 80 µl EtOH absolut dan 40 µl 5 M NaCl

13. Freezing selama Over Night (ON)

14. Sentrifuge pada kecepatan 12000 rpm selama 5 menit, buang bagian supernatan 15. Tambahkan 800 µl EtOH 70%

16. Sentrifuge pada kecepatan 12000 rpm selama 5 menit, buang bagian supernatan 17. Diamkan dalam keadaan terbuka (dalam desikator/ruang terbuka sampai alkohol

menguap/hilang)

18. Tambahkan 100 µl TE 80% atau Elution Buffer

Lampiran 3. Tabel Pengamatan dan Harapan Genotipe

Bangsa Populasi Genotipe

Bangsa Populasi Nilai Harapan Genotipe

Dalam dokumen Identifikasi Keragaman Gen Hormon Pertumbuhan Exon 4 pada Kambing PE, Saanen dan PESA dengan Metode PCR-SSCP (Halaman 75-86)