Saltingout extraction (SOE) based on lower molecular organic solvent and inorganic salt was considered as a good substitute for conventional polymers aqueous twophase extraction (ATPE) used for the extraction of some bioactive compounds from natural plants resources. In this study, the ethanolammonium sulfate was screened as the optimal SOE system for the extraction and preliminary purification of allicin from garlic. Response surface methodology (RSM) was developed to optimize the major conditions. The maximum extraction efficiency of 94.17% was obtained at the optimized conditions for routine use: 23% (ww) ethanol concentration and 24% (ww) salt concentration, 31 gL loaded sample at 25 C with pH being not adjusted. The extraction efficiency had no obvious decrease after amplification of the extraction. This ethanolammonium sulfate SOE is much simpler, cheaper, and effective, which has the potentiality of scaleup production for the extraction and purification of other compounds from plant resources
Trang 1~'~ECHNICAL BIPS Use of glycerol for enhanced efficiency and specificity of PCR a m p l i f i c a t i o n
by including digoxigenin-ll-dUTP during insert
amplification It is therefore important that probes PSR 123 PSR 135 PSR463 PSR 463 are efficiently amplified Here, we describe the use Glycerol (% ~ 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20
of glycerol to enhance the efficiency and specificity
of PCR amplification of fragments from cDNA and kt,
Attempting to use standard PCR conditions to 3.05
amplify inserts from clones isolated from wheat 1.64
of probes either failed to amplify or amplified 0.51
incorrectly (very low yield, fragment sizes different 0.z2
from those expected, etc.) Addition of 5% dimethyl
sulfoxide (DMSO) to the reaction buffer resulted
in efficient amplification of these probes (data •
not shown) Here, we report that the inclusion of
glycerol in the reaction buffer can also improve
PSR probes I are cloned in pUC18 vector Effect of glycerol concentration and annealing time on PCR yield and Representative results for two cD?'JA probes (PSR123 specificity of three representative PSR probes The concenta-ation of glycerol and PSR135) and one genomic probe (PSR463) in the reaction buffer is given at the top ~f each lane
are shown in Fig 1 Two pairs of oligonucleotides
homologous to pUC sequences flanking the inserts were used as primers: 5'-^rrc~mcrcc~'r^cc-y and 5'-Ac~'rCGACrCTA~^~;-y for the Sinai Inserts, PSR123 and PSR135; 5t-AACAGCTATGACCATG-3 t and 5'~a'^m, mG^Ct;GCC^GT-3' for the EcoRl insert, PSR463 PCR amplifications were performed in 50 1,1 of buffer: 10 mM TrisHCI pH 8.3, 1.5 mM MgCI 2, 50 mM KCI, 0.1 mg m1-1 gelatine,
200 gtM of each dNTP, 1 ~ of each primer, 1.25 U Taq polymerase (Boehringer Mannhein) 50 ng of plasmid DNA template were added to each reaction Glycerol was added to reactions at 5, 10, 15 or 20% of the final reaction volume Amplifications were carried out in a GeneAmp PCR system 9600 (Perkin Elmer Cetus) as follows: 1 min denaturation at 94°C, followed by
35 cycles of 15 s at 94°C, 30 s at 48°C, and 30 s at 72°C, and a final extension for 4 min at 72°C For probe PSR463, a second set of amplifications was performed using a decreased annealing time (20 s) 10 ill of each sample were analysed by electro- phoresis on a 1% agarose gel, and stained with ethidium bromide
When standard PCR buffer was used, no fragmem was amplified from PSR123, and instead of the expected 1 kb fragment,
a -2 kb fragment was amplified from PSR135 This was observed even when denaturation and/or extension times were increased Addition of 5%0 of glycerol to the reaction buffer resulted in efficient amplification of the correct fragment from PSR123 Amplification of PSR135 sequences yielded only a small quantity of the desired fragment, together with an abun- dance of the incorrect -2 kb fragment; however, addition of 10% glycerol led to amplification of the expected fragment from this clone Under the same thermal cycling conditions, PSR463 sequences were steadily amplified in the standard reaction buffer However, when the annealing time was decreased from 30 s to 20 s, a fragment of only 300 bp, instead of the expected 1.5 kb fragment, was weakly amplified from PSR463 The presence of 5-20°6 of glycerol in the reaction buffer seems to offset the efti~ct of the decreased annealing time
The exact role of glycerol in the PCR reaction is unclear, although it may act to prevent the fbrmation of secondary structure during amplification, much in the same way as DMSO 2 Like DMSO, the presence of glycerol may affect the thermal activiW profile of Taq polymerase and/or the extent to which strand-separation of the amplified product is achieved at a particular 'denaturation' temperature The results observed with PSR463 suggest that glycerol may affect the To, of the primers
Although DMSO (5%) did not appear to inhibit the activity of Taq polymerase in this study or in that of Shen and Hohn 2, such inhibition has previously been repotted -~.4 DMSO is very toxic, while glycerol is harmless and is present in high concen- tration in all Taq storage buffers It therefore seems that glycerol is more suitable for use in enhancing PCR amplification of sequences that are not efficiently amplified under standard conditions
Addition of glycerol to the PCR buffer also increases the reaction yield for probes that amplify poorly under standard conditions, but in certain instances increasing the concentration of glycerol to 20% decreased the amount of product obtained We propose that 10% glycerol be routinely added to the PCR buffer to maximize efficient specific amplification
It is possible that amplification might be further improved by using longer oligonucleotide primers and/or a higher annealing temperature
ACKNOWLEDGEMENTS
We thank M.D Atkinson, M Bernard, P Leroy and R Dumas de Vaulx for critical reading of the manuscript PSR probes were kindly provided by M.D Gale, Norwich, UK
REFERENCES
1 Sharp, p.J Chao, S Desai, S and Gale, M.D (1989) Tbeor AppL Genet 78 342-348
2 Shen, W.H and Hohn, B (1992) Trends Genet 8, 227
3 Gelfand, D.H (1989) in PCR Technology: Principles and Applications for DNA Amplification ( Erlich, H.A., ed.),
pp 17-22, Stockton Press
4 Saiki, R.K (1989) in PCR Technology: Principles and Applicalionsfor DNA Amplification (Erlich, H.A., ed.), pp %16, Stockton Press
Contributed by Yun Hal Lu and Sylvie NOgre, Station INRA d'Amdlioration des Plantes 63039 Clermont-Fen°and France
TIG SEPTEMBER 1993 V O L 9 xo 9
m