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

200


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
(A) 0.10
Absorbance
0.05

0.00
–0.05
300 400 500 600 700
Wavelength (nm)

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