14-01-2013, 04:53 PM
Optimization of DNA Extraction Methods for Some Important Forest Tree Species
ABSTRACT
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DNA quantity, extracted by five different methods, significantly varied among the six tree species investigated .Fresh leaves of B. serrata yielded maximum amount of DNA with overall mean of 1614.24 μg g-1 fresh leaf followed by A. procera with overall mean of 1357.24 μg g-1 fresh leaf. There was not much variation in quantities of DNA extracted from leaves of D. sissoo, G. arborea, J. curcas and P. pinnata (overall mean approx. 1000 μg g-1 fresh leaf) .Variation among samples of different tree species may be due to either intrinsic variation in terms of number and size of actively dividing cells or size of genome.
Extraction methods significantly influenced DNA quantity from the tree species investigated .The method of Lin et al. (2001) yielded maximum amount of DNA with overall mean of 1898.33 μg g-1 fresh leaf followed by the method of Cheng et al (1997) (overall mean = 1613.50 μg g-1 fresh leaf). DNA quantity was minimum in the method of Doyle and Doyle (1987) with overall mean of 444.44 μg g-1 fresh leaf .
Variation among extraction methods could be possibly due to varied composition of extraction buffers, varied components and parameters for precipitation and purification of DNA. For example, the method of Lin et al. (1997) uses SDS buffer for DNA
extraction and comparatively few steps for completion of the entire extraction process. On the contrary, the method of Doyle and Doyle (1987) involves several time consuming extraction steps.
DNA quality (or purity) was examined by recording the absorbance of DNA preparations at 260 and 280 nm and computing A260:A280 ratio. A260:A280 ratio of more than 1.8 indicates high quality whereas values less than 1.8 indicate protein contamination. DNA extraction methods and tree species were significant sources of variation for quality of extracted DNA. Of the extraction methods tested, those of Bousquet et al, Michiels et al. and Cheng et al. yielded DNA of the highest quality with spectrophotometer absorbance ratio (A260:A280) of 1.76, 1.69 and 1.65 respectively .Doyle and Doyle method resulted in the lowest quality of DNA with absorbance ratio of 1.55. However, with this method, except for B. serrata and D. sissoo, which yielded DNA with high quantity of protein contamination, other trees yielded high quality DNA with absorbance ratio of about 1.7. Among the tree species, G. arborea, J. curcas and A. procera consistently yielded DNA with high purity ratio (A260:A280 ≥ 1.8) with all the five methods tested. However, B. serrata yielded DNA with the lowest purity ratio (overall mean of 1.37) .
Variation in quality of DNA can be of two reasons: (1) Intrinsic ontogenetical, structural and biochemical variation among leaf samples of different tree species or (2) Variation in types of buffers used for extraction as well as the different steps of extraction with varying parameters and chemicals/reagents.
Restriction enzyme treatment of samples from all the tree species using all the five methods showed considerable amount of digestion .
Time and cost associated with DNA extraction and purification methods greatly influence marker associated studies, fingerprinting and genome mapping (Weising et al. 1995). The present study showed that there was variation in time required for different DNA extraction methods (please see Materials and Methods). The method of Lin et al. and Cheng et al. consisted of comparatively few steps for completion of the entire extraction process and were the most rapid extraction methods requiring less than three hours. On the contrary, the method of Bousquet et al., Cheng et al., Doyle and Doyle and Michiels et al. involved several time consuming extraction steps and took more than 16 hours to finish the entire processes.
A major research focus in molecular marker studies is optimization of different parameters for PCR analysis (Weeden et al. 1992; Wolf et al. 1993; Staub et al. 1996). Quality and quantity of DNA are critical factors in molecular marker studies. Present study showed that DNA extraction methods and tree species influenced ISSR amplification of extracted DNA. Among the five methods investigated, only the method of Bousquet et al. extracted amplifiable DNA from all the six tree species. This method also produced DNA with high purity ratio. The methods of Cheng et al., Doyle and Doyle and Michiels et al. could extract amplifiable DNA from A. procera, D. sissoo, G. arborea, J. curcas and P. pinnata. Failure of DNA amplification from samples of B. serrata using these methods may be explained by the low purity ratio of these DNA samples indicating protein co-precipitation of extracted DNA. With reference to the method of Lin et al., DNA samples extracted through this method from A. procera and G. arborea only were amplified as shown in the ISSR assay . This method extracted DNA with very low purity from B. serrata (1.28), P. pinnata (1.47) and D. sissoo (1.68) which
could possibly the reason for absence of DNA amplification in these samples .However, reason for failure of DNA amplification from samples of J. curcas which had high purity ratio (1.82), is not clearly understood. It is possible that such samples, even with high purity ratio, may still have trace levels of co-precipitation of phenols or other secondary metablolites, which could not be removed by the extraction method like that of Lin et al. It is important to note that all the other DNA samples from this species extracted by the other methods showed ISSR amplification.