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Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants

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Title Rapid high throughput template preparation (rHTTP) method: a novel cost effective method of direct PCR for a wide range of plants
Not Available
 
Creator Prassan Choudhary
Sudipta Das
Hillol Chakdar*
Arjun Singh
Arjun Singh
Sanjay Kumar Goswami
Anil Kumar Saxena
 
Subject Plant
DNA
high throughput
PCR
SSR
 
Description Not Available
Background: Conventional plant DNA isolation methods are complex, time consuming and require technical
expertise. These limitations were overcome using the DNA isolation kits which, however significantly add to the
research costs. Hence the present study was aimed to develop a high throughput, rapid and inexpensive method
of PCR ready DNA template preparation from plant materials.
Methods: Concentration of SDS in lysis buffer, amount of starting material, period and temperature for lysis were
optimized for obtaining PCR ready templates from plant materials. The method was tested using RAPD and ITS
specific primers for different plant species like rice, wheat, mustard, pea, soybean, pigeonpea, tomato, maize, march
lilly, bougainvillea, Indian blanket flower, nerium, petunia, purple pirouette petunia, moses-in-the-cradle, golden
cane palm, duranta, periwinkle, chrysanthemum and two xerophytes viz. Dipterygium glaucum and Crotaleria burhia.
SSR markers RM18398 and RM26108 showed successful amplification in rice varieties Improved Pusa Basmati 1 and
KS Dev 12. The effectiveness of the method was tested using fresh as well as 1 year old tissues. The storability of
the lysate was also tested.
Results: In this report, we developed a novel method called rapid high throughput template preparation (rHTTP)
method to prepare PCR ready DNA templates. Most striking feature of this technique is that it can be done
anywhere where water can be boiled by any means. Using rHTTP method, PCR ready templates can be prepared in
just 10 min. Robust and reproducible amplification for all the test plants were recorded with RAPD, plant ITS primers
and SSR markers following this method. rHTTP methods works well for both fresh as well as old plant tissues. The
lysates had a shelf life of 1 month when stored at 4 °C and 3 days when stored at room temperature.
Conclusions: rHTTP method has several advantages over the other protocols like ease of execution, no
requirement of tissue grinding/liquid nitrogen/hazardous chemicals and above all, equally effective for both fresh
and old samples. Using this method, costs per prep comes down ~ 10–50 times as compared to most commercial
kits. This method can be used for on-field experiments like molecular diagnostics, varietal identification etc.
Not Available
 
Date 2021-07-31T04:27:39Z
2021-07-31T04:27:39Z
2019-10-26
 
Type Research Paper
 
Identifier Not Available
Not Available
http://krishi.icar.gov.in/jspui/handle/123456789/51582
 
Language English
 
Relation Not Available;
 
Publisher Not Available