USING THIS ARTICLE LINK: http://www.scielo.br/scielo.php?script=sci_arttext&pid=
ID: 148871 • Letter: U
Question
USING THIS ARTICLE LINK: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0001-37652015000200691&lng=en&tlng=en
Answer as many questions as you can, if you can't answer all. I understand.
1- What is the major idea of the paper? Which technique gave the best result? (2 points)
2- How did they get rid of the soil in the samples (before testing the 4 techniques)? Were they able to get rid of all the soil in the samples (again before testing the 4 techniques)? (3 points)
3- What did they use (enzymatic, chemical or physical or all combined) to extract DNA from the samples in EACH procedure. (12 points)
4- When the DNA was extracted some methods did not lead PCR products. Why was that? (3 points)
Explanation / Answer
1) Main Aim of this research work was to s get standardized a protocol for isolating DNA from Nematode in the soil, such that they can use this method for a large amount of soil which will be containing nematodes from different categories. They have used four techniques using Tris/EDTA, Sodium dodecyl sulfate, Chelex 100 resin and using CTAB. Among these four techniques, the CTAB method combined with proteinase K and Chloroform-isoamyl alcohol gave the best results.
2) to get nematodes from soil samples they have used the centrifugal-flotation method. They couldn`t completely remove soil from samples. Because they have mentioned that they couldn`t get amplification in some DNA extraction methods were inefficient because of coextraction of inhibitors from the soil.
3) In the experiments, they have used physical disruption of nematodes combined with chemical extraction and /or enzymatic lysis a
) Method using TE buffer: DNA is isolated using physical disruption in TE buffer
b) Method using sodium dodecyl sulfate. They have used chemical lysis followed by chemical extraction of DNA using chloroform.
c) Method using chelex resin: they used physical disruption in TE Buffer to isolated DNA
d) Method using CTAB: They used physical disruption followed by extracting DNA using chemicals chloroform-isoamyl alcohol, extracted DNA was then precipitated using Isopropanol.
4) Methods other than CTAB couldn`t give amplification in PCR because in those methods inhibitors of the Taq polymerase were also extracted from the sample along with DNA. In the CTAB method, humic acids were removed from DNA humic acids can inhibit enzymes in PCR.
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