Chem 8157

Here are my questions and comments for Jon and the rest of the class.

1. Methodology - How does the device to conduct the PCR reactions was built? It seems that they will have problems with evaporation and mixing if the total volume per well is one nanoliter. Earlier references or a visit to the relevant company's web site may be needed.

2. Methodology -What are the pro's and con's of using one-step or two-step PCR?

3. Methodology - There are several panels in Figure 1. What does each panel represent? A different sample, a different preparation?

4. Data interpretation- See the last statement in page 7578. "In this manner, one can achieve nearly arbitrary sensitivity simply by increasing the scale of the assay". By looking at Figure 3, 10% trisomy, I do not necessarily see how sensitivity will increase by adding more compartments? Can someone explain or comment?

In response to Edgar’s first question, I went to the company’s website and found the design for the chip and for the device the conducts the PCR reactions. The websites are as follows:
Chip:
http://www.fluidigm.com/ProductInfo/DigitalArray.pdf
PCR machine:
http://www.fluidigm.com/biomark_absolute_quantification_instruments.htm#bm
They are vague about how they address some of the problems mentioned in the earlier post, however what I gathered from the literature is that wells that the samples are put into are sealed after the sample has been injected by the machine so that evaporation cannot occur. Also from checking out the company’s web site it appears that quantification of the GAPDH gene is how the company shows the reliability of their chip, which is probably why the authors of the paper decided to use that gene as their control. If people have more questions about the instrumentation I would recommend checking the site out.

The advantages to one-step PCR:
1) easier to set up
2) less expensive
3) less handling of products, therefore less introduction of error

Disadvantages of one-step PCR:
1) everything occurs in one tube - so no other genes can be amplified in later analysis
2) there is no way to save RNA for future runs

Advantages to two-step PCR:
1) random primers are kept in a separate tub, so all of the RNA can be converted to cDNA and can then be saved for later use without ruining the sample

In response to Edgar’s 3rd question, I do not think that there is a difference is the panel methodology between the same samples. In the top half of figure 1 the authors are looking at how many copies of chromosome 21 there are. The left half looks at the DNA taken from a normal cell line whereas the right half looks like it is taken from a trisomy 21 cell line. The bottom panel is looking at how much of the GAPDH gene there is and again the left half is wild type DNA and the right part is trisomy 21.
I believe the reason that they run so many multiple panels is because in order to get a reliable ratio they need thousands of test samples. They only way they could accomplish this is by running multiple lanes. From what I read in the company’s website it sounded like they had already idealized the conditions in which to run the PCR reactions for the chips. It would be interesting to see if the authors tried to tweak those conditions for there PCR reaction, but I did not see that information given in the paper.

The anonymous poster is actually me. I don't know why it tagged me as anonymous.