Czech J. Food Sci. Vol. 27, 2009, S

Czech J. Food Sci. Vol. 27, 2009, Special Issue 2: S2-75–S2-81





(bp)

500

300

211

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Figure 4. CTAB extracted DNAs from papaya flesh and stone – amplification of specific region of internal pa-pain gene (211bp) is indicated by arrow. Line contents: M – HindIII DNA ladder (Fermentas, Canada); 1 – posi-tive control; 2 – papaya flesh (isolation A); 3 – papaya flesh (isolation B); 4 – papaya stone (isolation A); 5 – papaya stone (isolation B); 6 – extraction control; 7 – MasterMix control

DNA quantity and quality are considered a basic pre-requisite for successfull PCR amplification (Hübner et al. 1999). A wide range of DNA concentrations can be used for qualitative PCR, quantitative PCR must use a pure DNA within a quite narrow concentration range to reach the treshold around Ct = 22 (Joint Research Centre 2004; Yun et al. 2006; Shokere et al. 2009). Otherwise DNA target gene relative concentration can not be calculated reliably (Bustin 2000).

Only DNAs isolated by CTAB based method from both of papaya fruit tissues (flesh and stone) were amplificable (Figure 4). Other methods of DNA isolation did not lead to amplifiable product.

No detectable amount of DNA was isolated by CTAB or Wizard based method from candied papaya as it was shown by specrophotometry and electro-phoretic separation. No amplification by PCR was recorded when aliquotes of resulting solution were used as templates. Manufacturer’s protocol recom-mended for GeneSpin DNA Isolation Kit did not lead to isolation of amplificable DNA either.

Candied fruit is processed by drenching in syrup, which made the fruit to be saturated with sugar. The high sugar content probably caused that the sample mixed with lytic buffer became viscous and difficult to separate.

Therefore, manufacturer’s protocol was modified. The volume of lytic buffer was increased up to 700 µl due to consistency of resulting supernatant. The time of incubation in the buffer was increased up to 1 h along with. Resulting DNAs were not de-tectable by gel electophoresis. Spectrophotometry showed presence of large mass of a staff absorbing over estimated range (Figure 5).







0.11
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Figure 5. Absorption spectrum of GeneSpin isolated DNA from the candied papaya

Also amplification did not occurred. Oguchi et al. (2009) reported absence and/or amplification inhibition of sugarbeat internal gene in extracts from different kinds of sugar. Also Ricaut et al. (2005) reported inhibition of PCR reaction by Maillard reaction products. To estimate possible presence of inhibitors, DNAs were diluted 10× and after that the reaction run over (Figure 6).

Amplifiable DNAs were tested for the presence of screening element CaMV 35S promoter as it is a part of transgene and the sequence is often for preliminary verification of transgene presence (Hübner et al . 1999) and it is involved in transgenic constructs of GM papaya (AgBios GM Database). As the detection limits of papain specific assays and CaMV 35S promoter assays are comparable (LOD = 0.05 ng), the approach can be employed for the first examination of products on the mar-ket. Although reference sample gave a positive



(bp)

500

300

211

200

Figure 6. GeneSpin extracted DNAs from candied papaya

– amplification of specific region of internal papain gene (211 bp) is indicated by arrow. Line contents: M – HindIII DNA ladder (Fermentas, Canada); 1 – positive control, 2 – candied papaya (isolation A); 3 – candied papaya (iso-lation B); 4 – extraction control; 5 – MasterMix control

Hasil (Bahasa Indonesia) 1:

Ceko J. makanan Sci. Vol. 27, 2009, khusus edisi 2: S2-75-S2-81 (bp)500300211200Gambar 4. CTAB DNAs diekstrak dari daging pepaya dan batu – amplifikasi wilayah tertentu dari gen pa-sakit internal (211bp) ditunjukkan oleh panah. Garis isi: M-HindIII DNA tangga (Fermentas, Kanada); 1 – kontrol loterei-tive; 2-pepaya daging (isolasi A); 3-pepaya daging (isolasi B); 4-pepaya batu (isolasi A); 5 – pepaya batu (isolasi B); 6 – ekstraksi kontrol; 7-MasterMix kontrolDNA kuantitas dan kualitas dianggap dasar pra-syarat untuk sukses PCR amplifikasi (Hübner et al. 1999). Konsentrasi berbagai rentang DNA dapat digunakan untuk kualitatif PCR, PCR kuantitatif harus menggunakan DNA yang murni dalam kisaran konsentrasi agak sempit untuk mencapai treshold di sekitar Ct = 22 (Joint Research Centre 2004; Yun et al. 2006; Shokere et al., 2009). Sebaliknya DNA target gen konsentrasi relatif tidak dapat dihitung terpercaya (Bustin 2000).Hanya DNAs terisolasi oleh CTAB berdasarkan metode dari kedua buah pepaya jaringan (daging dan batu) yang amplificable (gambar 4). Metode lain isolasi DNA tidak mengarah ke amplifiable produk.Tidak ada jumlah terdeteksi DNA terisolasi oleh CTAB atau Wizard berbasis metode dari manisan pepaya seperti yang ditunjukkan oleh specrophotometry dan pemisahan elektro-phoretic. Amplifikasi tidak oleh PCR direkam ketika aliquotes dari solusi yang dihasilkan digunakan sebagai template. Produsen protokol recom-diperbaiki untuk GeneSpin DNA isolasi Kit tidak mengarah ke isolasi DNA amplificable baik.Manisan buah-buahan yang diproses oleh membasahi dalam sirup, yang membuat buah jenuh dengan gula. Kadar gula tinggi mungkin disebabkan bahwa sampel dicampur dengan litik penyangga menjadi kental dan sulit untuk memisahkan.Oleh karena itu, produsen protokol diubah. Volume litik penyangga meningkat hingga 700 μL karena konsistensi supernatant dihasilkan. Waktu inkubasi dalam buffer meningkat sampai dengan 1 h bersama. Dihasilkan DNAs itu tidak de-tectable oleh gel electophoresis. Spektrofotometri menunjukkan kehadiran massa yang besar dari staf menyerap selama rentang yang diperkirakan (gambar 5). 0.11 (A) 0,10 Absorbansi 0,05 0.00 –0.05 300 400 500 600 700 Panjang gelombang (nm) Gambar 5. Penyerapan spektrum GeneSpin terisolasi DNA dari manisan pepayaJuga amplifikasi melakukan tidak terjadi. Oguchi et al. (2009) melaporkan adanya dan/atau penghambatan amplifikasi gen sugarbeat internal di ekstrak dari berbagai jenis gula. Juga Ricaut et al. (2005) melaporkan penghambatan PCR reaksi oleh produk Reaksi Maillard. Untuk memperkirakan tersedia hadapan inhibitor, DNAs diencerkan × 10 dan setelah itu reaksi menabrak (gambar 6).Amplifiable DNAs diuji untuk kehadiran skrining elemen CaMV 35S promotor seperti itu adalah bagian dari transgene dan urutan sering untuk verifikasi awal kehadiran transgene (Hübner et al. 1999) dan itu adalah terlibat dalam konstruksi transgenik GM pepaya (AgBios GM Database). Sebagai deteksi batas papain khusus lengkap dan CaMV 35S promotor tes sebanding (LOD = 0.05 ng), pendekatan yang dapat digunakan untuk pemeriksaan pertama produk di mar-ket. Meskipun referensi sampel memberikan positif(bp)500300211200Gambar 6. GeneSpin diekstrak DNAs dari manisan pepaya-amplifikasi wilayah tertentu dari internal papain gen (211 bp) ditunjukkan oleh panah. Garis isi: M-HindIII DNA tangga (Fermentas, Kanada); 1-kontrol positif, 2-manisan pepaya (isolasi A); 3-manisan pepaya (iso-hal yang sanggat menarik B); 4 – ekstraksi kontrol; 5-MasterMix kontrol