Name: rapid high throughput amylose determination in freeze dried potato tuber samples
Uploaded: 2013-10-14T21:00:00
Description: Watch this Scientific Journal Video about Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples at JoVE.com

Funding for this research was provided in part by the USDA-ARS Research Associate Program and the USDA Crop Germplasm Committee.

1. Amylose Determination from Potato Tuber Starch
<ol>
	<li>Peel and cut fresh tubers into small cubes.</li>
	<li>Place potato cubes in small brown bags and store overnight at -80 &#176;C.</li>
	<li>Transfer potato cubes to nylon bags and for freeze drying.</li>
	<li>After freeze dried, grind the potato sample into powder using mortar and pestle or a Wiley Mill.</li>
	<li>In a 50 ml tube, add 20-30 mg of the freeze-dried, ground tuber sample.</li>
	<li>Prepare a solution of 45% (w/v) perchloric acid (mix 24.4 ml of 60%...

<ol>
	<li>Composition Hoover, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=molecular+structure,+and+physicochemical+properties+of+tuber+and+root+starches:+a+review.">molecular structure, and physicochemical properties of tuber and root starches: a review.</a> Carbohydr. 45 (3), 253-267 (2001).</li><li>Bertoft, E., Blennow, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Potato+Starch.">Structure of Potato Starch.</a> Advances in Potato Chemistry and Technology. , 83-98 (2009).</li><li>Campbell, M. R., Yeager, H., Abdubek, N., Pollak, L., Glover, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+methods+for+amylose+screening+in+maize+starches+from+exotic+backgrounds.">Comparison of methods for amylose screening in maize starches from exotic backgrounds.</a> Cereal Chem. 79, 317-321 (2002).</li><li>Zhu, T., Jackson, D. S., Wehling, R. L., Geera, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+amylose+determination+methods+and+the+development+of+a+dual+wavelength+iodine+binding+technique.">Comparison of amylose determination methods and the development of a dual wavelength iodine binding technique.</a> Cereal Chem. 85, 51-58 (2008).</li><li>Hovenkamp-Hermelink, J., Devries, J., Adamse, P., Jacobsen, E., Witholt, B., Feenstra, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+estimation+of+the+amylose+amylopectin+ratio+in+small+amounts+of+tuber+and+leaf+tissue+of+the+potato.">Rapid estimation of the amylose amylopectin ratio in small amounts of tuber and leaf tissue of the potato.</a> Potato Res. 31, 241-246 (1988).</li><li>Shannon, J. C., Garwood, D. L., Boyer, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+Physiology+of+Starch+Development.">Genetics and Physiology of Starch Development.</a> Starch. , 23-82 (2009).</li><li>Haase, N. U., Plate, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+potato+starch+in+relation+to+varieties+and+environmental+factors.">Properties of potato starch in relation to varieties and environmental factors.</a> St&#228;rke. 48 (5), 167-171 (1996).</li><li>Johnston, F. B., Urbas, B., Khanzada, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+storage+on+the+size+distribution+and+amylose/amylopectin+ratio+in+potato+starch+granules.">Effect of storage on the size distribution and amylose/amylopectin ratio in potato starch granules.</a> Am. Pot. J. 45, 315-321 (1968).</li><li>Gibson, T. S., Solah, V. A., McCleary, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+to+measure+amylose+in+cereal+starches+and+flours+with+concanavalin+A.">A procedure to measure amylose in cereal starches and flours with concanavalin A.</a> J. Cereal Sci. 25 (2), 111-119 (1997).</li><li>Sun, S., Matheson, N., Yun, S. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+amylose+content+of+starches+after+precipitation+of+amylopectin+by+concanavalin+A.">Estimation of amylose content of starches after precipitation of amylopectin by concanavalin A.</a> Starch. 42, 302-305 (1990).</li></ol>

Amylose content in potato tubers typically ranges from 20-35%7,8. If most values are outside this range, consider these possible errors: 1) raw data were used instead of blanked data for the construction of the amylose/amylopectin standard curve; 2) raw data were used instead of blanked data for the determination of the amylose/amylopectin ratios from the potato samples; or 3) the water used for the analysis was not pure enough. We noticed that by using distilled water and not ultra-pure water in the analysis,...

Approximately 80% of the fresh weight of a potato tuber is water; nearly all of the remaining dry matter is starch1. Most of the starch (70%) is composed of amylopectin, while the remainder is amylose. The ratio between amylose and amylopectin is the most important property influencing the physical properties of starch. Amylose is a linear alpha 1-4 glucose chain, while amylopectin is a linear alpha 1-4 chain with alpha 1-6 branches2. Methods such as iodine binding, differential scanning calorimetry (DSC), high performance size exclusion chromatography (HPSEC) and concanavalin A interactions have been developed for starch determination in different crop species3,4. Each protocol requires special skills and equipment, making it difficult to scale up to quantify numerous samples at a time. The method we present here is a modified protocol from Hovenkamp-Hermelink et al.5 which is a dual wavelength iodine binding method based on stained starch granules. The advantages of this method versus others include the amylose determination from raw starch without purifying it and the use of the dual wavelength system to increase the precision of the method4,6.&#160;

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td> Reagents</td>
 </tr>
 <tr>
 <td>Amylose </td>
 <td>Sigma</td>
 <td>859656</td>
 <td></td>
 </tr>
 <tr>
 <td>Amylopectin </td>
 <td>Sigma</td>
 <td>A8515</td>
 <td></td>
 </tr>
 <tr>
 <td>Perchloric Acid 60%</td>
 <td>Sigma</td>
 <td>311413</td>
 <td>Very hazardous to skin and eyes</td>
 </tr>
 <tr>
 <td>Iodine</td>
 <td>Sigma</td>
 <td>23214TD</td>
 <td></td>
 </tr>
 <tr>
 <td>Potassium iodide</td>
 <td>Sigma</td>
 <td>08625JE</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Plate Reader</td>
 <td>Bio Tek</td>
 <td>ELx800</td>
 <td></td>
 </tr>
 </tbody>
</table>

rapid high throughput amylose determination in freeze dried potato tuber samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

The standard curve constructed from blanked data of different amylose/amylopectin concentration solutions shows approximate absorbance ratios ranging from 0.7 to 1.6 (Table 1). A linear regression trend line from these data is later used for inferring amylose content in freeze dried potato samples (Figure 1).
The plant production environment has some effect on the amylose content in the potato tuber7. Replicated amylose content determinations in two...

Watch this Scientific Journal Video about Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples at JoVE.com

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

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