‘Touch-incubate-PCR’ Approach (aka fasTip-X Kit) for High Throughput Genotyping: System 1
The fasTiP-X approach is a rapid extraction method which allows direct amplification without going through conventional CTAB extraction. In the same time, it allows DNA extraction without contacting any harmful chemicals and liquid nitrogen. This method offers a great advantage whereby it requires only approximately 20 minutes for DNA preparation before PCR amplification thus increases the possibility for high-throughput genotyping. Apart from that, the requirement of small amount of plant material is greatly suitable for samples with limited quantity.
The initiative for developing this approach was due to the common hindrance faced by the researchers where sample collection and preparation was the most time consuming part of the project. Hence, this approach was developed to reduce the time, cost and consumable volume required for sample DNA extraction. This rapid DNA extraction approach only involves 3 simple steps before PCR amplification, which is: 1) transferring plant samples into the extraction buffer by touching the sample leaves by using pipette tips or Harris Uni-CoreTM puncher 0.5 mm; 2) incubate to lyse plant cells, and 3) the incubated solution can be directly used for PCR amplification. However, care must be taken if Harris Uni-CoreTM puncher is used for obtaining samples. A 2% solution of sodium hypochlorite (NaCIO) shall be used for cleaning the puncher to prevent cross-contamination.
The fasTiP-X approach was tested using 5S rRNA primers via PCR on 4 different species, namely Neolamarckia cadamba (Roxb.) Bosser (Kelampayan), Duabanga moluccana (Sawih), Durio zibelthinus (Durian) and Dimocarpus longan Lour. (Longan). The amplification of DNA template obtained from the fasTiP-X was comparable to the positive control which extracted using conventional CTAB method. The PCR analysis using DNA template isolated by the fasTiP-X approach for each species was repeated 3 times to prove the reliability and reproducibility of this method. This result showed that the fasTiP-X approach has higher potential for high-throughput genotyping compared to the conventional DNA extraction by considering its rapidity, simplicity and cost-effective features.
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