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I am trying to devise a quick method to extract genomic DNA from human cells for a PCR.

I first collected cells by centrifuging saline mouthwash (0.9% NaCl) and extracted genomic DNA using kit followed by a PCR protocol which works fine but is a rather lengthy process.

I tried to cut short the method and removed the DNA extraction steps by using pelleted spit samples as templates for PCR and increased the initial denaturing step from 3 minutes to 5 minutes. However this method was not reliable as some of my samples were amplified and some were not.

Can anyone suggest me a reliable protocol here?

Thanks!

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  • $\begingroup$ Why do you need to shorten the protocol? $\endgroup$ – MattDMo Nov 10 '15 at 14:53
  • $\begingroup$ I am trying to make a protocol in which DNA extraction, PCR and gel electrophoresis could be done in a time frame of about 4-5 hrs and I am trying to compact everything. $\endgroup$ – techtrix Nov 10 '15 at 18:22
  • $\begingroup$ @techtrix I am curious, did you try the boiling method? $\endgroup$ – Ro Siv Nov 17 '15 at 1:40
  • $\begingroup$ @RoSiv not yet. I may start my work next week. I will let you know how it turns out. $\endgroup$ – techtrix Nov 17 '15 at 11:21
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I am not confident in my reply, but the article linked below has methods for DNA extraction of various human sample types( blood, hair, urine, Cheek)

For what its worth, my professors always told me DNA extraction was more of an art than a science, and one has to tinker to get it just right.

Maybe for something really quick, you could try a protienase K "squishing buffer" and then boil it for like 10 mins in the buffer and then try running PCR. I am just an undergraduate though so take my words with a heavy grain of salt.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3792701/

Here is how they gathered the buccal samples: buccal swabs:

five healthy adult volunteers were recruited (age range, 22–35 years), and demographic information, including age, health condition, sex, population ancestry, hair color, and hair treatments, was collected. The volunteers recruited were asked to rinse their mouth with tap water, 30 s before sampling of buccal swabs, to avoid the contamination as a result of food particles. For each individual, both sides of buccal mucosa were wiped with a cotton swab for 15 s, and a total of five samples was collected in 500 μl 10 M Tris-HCl, 10 mM EDTA, 2% SDS, containing 1.5-ml microcentrifuge tubes. Isolation of DNA from cotton swabs was performed.

Here is a copy paste, hyphens are mine.

DNA Extration:

Buccal swabs:

-samples were suspended in 500 μl lysis buffer [10 mM Tris (pH 8.0), 10 mM EDTA, and 2.0% SDS], and 50 μl 10% SDS, followed by 5–10 μl 20 mg/ml proteinase K

-incubated 1–3 h at 56°C until the tissue was totally dissolved

-The DNA was then extracted from each sample with an equal volume of phenol:chloroform: isoamyl alcohol solution (25:24:1) and mixed gently by inverting the tubes for 3 min.

-samples were then centrifuged (Eppendorf 5415R; Hamburg, Germany) for 10 min with 10,000 g (4°C),

-upper aqueous layer was transferred to a fresh, sterilized microcentrifuge tube.

-RNase A (10 μl of 10 mg/ml; Fermentas, Thermo Scientific, Germany) was added, and the solution was incubated at 37°C for 30 min.

-Equal volumes of chloroform:isoamyl alcohol solution were added and centrifuged (Eppendorf 5415R), again with 10,000 g (4°C) for 10 min.

-The upper aqueous layer was transferred to a sterilized microcentrifuge tube, and double the volume of chilled isopropanol (Merck, Whitehouse Station, NJ, USA) was added, along with one-tenth volume of 3 M sodium acetate, and chilled at −20°C for 1 h for precipitation.

-After 1 h, the sample was centrifuged (Eppendorf 5415R) at 10,000 g (4°C) for 10 min.

-After decanting the supernatant, 250 μl 70% ethanol (Merck) was added, and the pellet was dissolved; the mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant was decanted gently.

-The pellet was air-dried under laminar air flow, and the dried pellet was resuspended in 50 μl nuclease-free water or 1× 10 mM Tris-HCl, 1 mM EDTA, pH 7.6 (TE), buffer and frozen at −20°C or at −80°C for storage.

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  • $\begingroup$ Thanks Ro Siv, but this process seems rather lengthy for me. I could try the protienase K "squishing buffer" method though. $\endgroup$ – techtrix Nov 10 '15 at 18:24
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I do a lot of gDNA and cfDNA extraction! What Ro Siv has explained is a pretty standard method of buccal swab extraction, however the pK incubation times do not need to be nearly that long. The gold standard in DNA extraction is QIAGEN kits. Specifically for your uses I would go with something like the QIAamp DNA Mini Kit (you can view their handbook protocols on their site - https://www.qiagen.com/us/resources/resourcedetail?id=67893a91-946f-49b5-8033-394fa5d752ea&lang=en) In practice, I have been able to do 4 samples without automation on their spin protocol in around 1.5 hours total.

PCR time will depend on your cycling conditions and how long it takes you to set up a reaction etc, and gels would depend on how precise you need your bands to be. I think the whole thing can easily be done in 4-5 hours. In my experience, blood samples take only slightly longer, but is obviously more invasive and less practical. I agree that buccal is the way to go. For reference, we use Puritan hydraflock swabs, but that's up to you.

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