Good day house. I have issues extracting high quality DNA from Blood spot on FTA paper for PCR and Sanger Sequencing.
1. Unclotted blood samples were spotted on FTA paper, and gDNA was extracted using QIAamp DNA mini kit. Gel electrophoresis confirm the presence of gDNA but the quality of the DNA and purity was terrible using NanoDrop Spec - while some read negative values many were less than 1 instead of > 1.6 What could have been responsible for the contamination as the manufacturer's instructions were followed to the letter?
2. PCR was performed using some primers, and Gel Electrophoresis conducted. From the gel, some of the samples were positive for the primers used (single band). These PCR positive samples were sent for Sanger Sequencing, but the company responded later that all the sent samples failed their Quality Control test, and requested we resend the amplicons. While trying to resend the amplicons, we discovered that there was nothing again (no more amplicons) in the PCR products.
What could have gone wrong despite using quality reagents for the extraction and the PCR?
Good morning Olayinka,
please have a look on the provided troubleshooting guide, Qiagen is highly specific for each sample source.
In your case, as you state that you were able to see your gDNA by gel electrophoresis, but with nano drop its quality was not reliable. This is usually obtained when you don´t prepare properly buffers AW1 and AW2, or that they are quite old. The quality of the ethanol used also affects the quality of you extractions. Even if Qiagen recommends using 96 to 100% ethanol, I suggest to use preferably 100% ethanol (of Molecular biology grade, to avoid any isopropanol or methanol in the mixture), as ethanol tends to evaporate even under cold climates. Ethanol evaporation, will also affect your sample, as you are not applying the proper chemistry to the extraction.
A bad gDNA extraction affects its suitability for PCR. Not having PCR products, on most of your samples, indicate that your protocol has a failure, and this is linked to the AW buffers.
As extra recommendation, if you're following the qiagen mini handbook, at step 8, reduce the centrifugation speed to 8000 rpm and increase from 3 min to 5 min. Step 9, is mandatory, as any residual ethanol will render you a good gDNA but of no quality. For step 9, change the 2ml collection tube (you can re-use some of the previously used if they are completely dry) and centrifuge for 5 minutes.
and finally, for the elution (step 10), use 100 ul of pure distilled water pre-warmed at 65C instead the buffer AE provided. Incubate at room temperature for 5-10 minutes, and then centrifuge as indicated at 8000 rpm for 2 minutes. You can re-do a second elution (in a new tube) to increase the amount of total gDNA obtained from the 3 spots. After verification, you can just merge both elution in one, but remember to re-measure its concentration with nano drop.
Dear Dr Okoh,
If you have sufficient ammount of sample, repurify it with the same Qiagen Kit. Often clinical samples are loaded with PCR inhibitors, which are not removed by single purification step. After repurification, perform a PCR with any host specific primer and see the difference. I hope this will improve your experiment.
good luck,
SD
Thanks very much Beatriz Sabater-Muñoz and Sibnarayan Datta . But can FTA Classic cards for any reason(s) compromise the integrity of a sample? I mean is it possible that FTA card could be responsible for the issues raised?
Hi Olayinka Sunday Okoh,
no, I don't think FTA classic cards will compromise the integrity of the sample. They have been developed for protecting samples for years!
What preservative you use to avoid blood to clot? Some blood preservatives are inhibitors of PCR reactions, but having a purification method, if properly applied, should help getting rid off these inhibitors. Unless the wash buffers are not properly stored.
hope this helps,
Beatriz
Beatriz Sabater-Muñoz I used EDTA to prevent clotting. The was first collected in EDTA sample container, mixed and then spotted on FTA cards.
With the FTA cards you don´t need to use EDTA, simply spot the blood into it. FTA cards contains already EDTA and other agents, to preserve your sample.
EDTA is a strong agent that inhibits PCR reactions, it is used below 0.1 mM to protect your sample while not-inhibiting the PCR reaction.
After saying this, if the washing buffers are properly prepared and no evaporation had occurred, should be enough to remove any excess of any salt.
For this I usually use PCR grade water (instead the elution buffer) to elute the gDNA from any extraction, specially for those that have been preserved.
If you are not able to repeat the extraction of your samples from other punches, I recommend to use more MgCl2 in your PCR reaction to improve yield. IF your samples still have a high amount of EDTA, using extra MgCl2 will improve the success of the PCR and any downstream application.
Hi, Olayinka Sunday Okoh
Let me ask you one thing that usually happens in tropical countries quite different from other places around the world.
What was your blank solution for the nanodrop evaluation? And how was it maintained/kept? Did you use the same solution used to elute your samples? Was this solution kept sterile? I am asking you that because sometimes people like to left an aliquote of elution buffer close to the nanodrop machine to do it faster and leave it for days (opening and closing it in the air). After sometime, bacteria and fungi starts to build up in the solution and then ... more DNA on the blanking solution than on your samples, giving negative results ...
An idea to be checked. Ok? Just get a new elution buffer or prepare a sterile TE solution and use it to blank and then measure your DNA samples.
Hope it helps.
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