Vol. 27, 2009, Special Issue 2: S2-

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



(bp) 23 130

9 416








Figure 2. CTAB isolated DNA from papaya fruit tissues flesh and stone. Lane contents: M – HindIII DNA ladder (Fermentas, Canada), 1 – DNA extracted from papaya flesh, 2 – DNA extracted from papaya stone, 3 – extraction control. The high-molecular DNA is indicated by arrow

provided by S. Pecoraro (Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany) was explored. IRMM (Institute for Reference Ma-terials and Measurements) reference materials of RoundUp Ready (RR) soya (GM line GTS 40-3-2) were used as positive controls for CaMV 35S pro-moter evaluation. Contents of CaMV 35S promoter equals to RoundUp ready contents (AgBios GM Database). Reference materials BF410a (≥ 0.03% RR soya), BF410b (0.1% RR soya) and BF410d (1% RR soya) were applied in each GM papaya screening PCR. Data were evaluated as recommended the ISO Standard (EN ISO 21571:2002).

Extraction control originated from DNA isola-tion – microtube without any sample for DNA isolation was treated in the same way as other samples. Extraction control serve to check of DNA contamination during isolation process.



MasterMix control (no DNA in reaction) was used for checking of contamination in PCR reagent.

RESULTS AND DISCUSSION

Usually, DNA isolation protocol affect quality and quantity of resulting DNA and possible use for downstream applications (Demeke et al. 2009). As it is not trivial to isolate pure DNA from all food matrices (Chapela et al. 2007; Murray et al. 2009), three different methods for DNA isolation (CTAB, Wizard method, GeneSpin DNA Isolation Kit) from two tissues of papaya fruit (flesh and stone) and from candied papaya were tested.

When extracts were analysed on 0.8% agarose gel by electrophoretic separation, that provides qualitative information on DNA degradation extent (Hübner et al. 1999), high molecular DNAs were not visible except for DNA isolated from papaya stone by CTAB procedure (Figure 2).

Spectrophotometry acknowledges the results showing low concentration of isolated DNA at or under the LOD of the instrument. Results of gel electrophoresis, measurement of DNA quantity and the amplifiability of DNA are summarised in Table 2. As low DNA concentration at detec-tion limit of the apparatus were reached, values reporting DNA purity (A260/A280) were not finally calculated.

For more detailed information, the absorption spectrum from 190 nm to 760 nm was recorded. Relevant peaks at 260 nm were noted in samples isolated by CTAB procedure from fresh fruit flesh and stones only (Figure 3).


(a)


0.11
(A)
Absorbance 0.10
0.05

0.00


(b)



0.11
(A)
Absorbance 0.10

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



Figure 3. Absorption spectrum of CTAB isolated DNA from the papaya stone (a) and the flesh (b)