Thanks for all that responded to my query about the extraction of total gDNA from avian blood samples on FTA cards. Below is the original post followed by many useful replies. *****Original post***** Our lab has a collection of dried avian blood (nucleated red blood cells) samples on Whatman FTA cards. Some samples consist of only a small spot of blood, while others contain several drops of blood with the sample soaking through and visible on the back of the card. Rather than using a sample disk directly in a PCR, we are interested in extracting total genomic DNA to be used as a stock solution in downstream sequencing. I'm wondering if anyone has experience with this, and might be able to provide advice on types of kits/protocols used. Also, any input on DNA quality/yield, sample variation, punch tools, and sample disk diameter would be appreciated. *****Papers***** Smith lm & LA Burgoyne. 2004. Collecting archiving and processing DNA from wildlife samples using FTA databasing paper. BMC Ecology. 4:4. Borisenko AV et al. 2008. DNA barcoding in surveys of small mammal communities: a field study in Suriname. Mol. Ecol. Res. 8: 471-479. McClure Mc et al. In press. Extraction of DNA from FTA® cards for use on the Illumina iSelect BeadChip. Animal Genetics. (Whatman GenSolve Kit vs Phenol:Chloroform:Isoamyl alcohol extraction) Barbanera, F et al. In press. Human-mediated introgression of exotic chukar (Alectoris chukar, Galliformes) genes from East Asian into native Mediterranean partridges. Biol. Invasions. *****FTA Elute***** The new product from Whatman - 'FTA elute cards' - requires only water both for purification and elution, and is much more efficient. If you plan to sample avian blood later on, I would recommend switching to FTA elute. *****Hole punches***** In terms of punches - the Harris Micro-Punch lasts forever and is a good investment: http://www.sigmaaldrich.com/labware/labware-products.html?TablePage=3D17216376 http://www.whatman.com/PRODFTAPurificationReagentandAccessories.aspx My preference is a 1.2 mm punch - if I need more DNA, I punch a card several times. This is also the optimal size to use the disk directly in PCR in a 12.5 or 25 ul reaction. Don't get Harris Unicore punches, they don't last! To ensure that your punch lasts, get self-healing Harris cutting mat: http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=3DZ708755|SIGMA&N5=Product%20No.|BRAND_KEY&F=SPEC *****Hole punches 2***** Oh, and for the punches, I simply bought the smallest available punch-out tool in the scrapbook department at Michael's (the craft store). *****Taq for PCR***** Regarding PCR... Platinum Taq from Invitrogen is one of the best enzymes to deal with samples from FTA cards. *****Qiagen DNeasy Kit***** We do this kind of work regularly. We have tried a number of extraction kits and organic extraction methods and now use only the Qiagen DNAeasy Tissue Kit. We use a small pair of dissecting scissors to cut out a small piece of the paper (and usually cut it up into pretty small pieces) because they can be cleaned between samples. The final step in the Qiagen kit is an elution in AE (or you can use TE or TLE depending on what you want the DNA stored in). This is one thing that can help control the DNA yield. We use 2 elutions, 100 ul of AE twice but you can change that to a 100 and a 50 ul elution to get higher concentrations. I suggest playing around a little with how much paper to use depending on the blood spot - our experience has been that even a very small spot works just fine - and so for very heavily soaked paper you may want to use less and/or elute with a higher volume of TLE, TE or AE buffer. We get consistently excellent yield (anywhere from 10 or 20 to >100 ng/ul) and the DNA is very good quality. We have used it primarily for microsatellite work but also for sequencing (mostly mtDNA) and for AFLPs. This is our working protocol for gauze and for paper (we tend to use filter paper - and have used other kinds of unlikely things - depends on the people in the field!) Everything not listed follows the manufacturer protocol for "Purification of Genomic DNA from Whole Nucleated or Non-nucleated Animal Blood," DNA was extracted from the blood on the gauze using a DNeasy Tissue kit (Qiagen®) following the manufacturer's protocol, "Purification of Genomic DNA from Whole Nucleated or Non-nucleated Animal Blood," with modifications. Rather than adding 5-10 µL anticoagulated blood to 20 µL proteinase K in the bottom of a 1.5 mL microcentrifuge tube, a sample of the gauze with a concentrated blot of blood was cut using sterile techniques and placed in a 1.5 mL microcentrifuge tube already containing the proteinase K. The circular piece of gauze was approximately 5 to 17 mm in diameter based on visual assessment of the concentration of the blood on the gauze. A volume of 190 µL of phosphate buffered saline (PBS) solution was added to adjust the total volume to approximately 220 µL. Two elutions were carried out using 100 µL of kit-provided buffer AE to create a final volume of 200 µL. The resulting DNA extract was stored at -20ºC until used for PCR. Clean scissors in 95-100% ETOH and wipe dry with kimwipe in between cuts. Use the same technique with filter paper.... *****Qiagen DNeasy Kit 2***** I've collected avian blood samples on Whatman filter paper and extracted genomic DNA for stock solutions. My protocol is adapted from one developed by Ken Petren (U Cincinatti), and is as follows: When collecting samples, I first add 1-2 drops of 0.5M EDTA (pH 8.0) to filter paper and allow paper to dry. After blotting blood onto the filter paper, I immediately add another 1-2 drops of EDTA, then allow the paper to dry somewhere shady. The samples can then be stored in plastic tubes containing a ball of drierite wrapped in cheesecloth. I've stored these bottles at 15-30C for up to 4 months before putting them in the -20C freezer. To extract the DNA, I just use a Qiagen DNEasy blood & tissue kit, adapting the protocol a bit to account for reagents being soaked into the filter paper: 1. cut a small piece of the blood sample off the filter paper and put in 1.5mL tube- needs to be small enough that it goes all the way down to the bottom of the tube. When cutting up samples, I dip the scissors in water and then ethanol, then flame the ethanol off between cutting each sample. 2. add 30ul Proteinase K, 190ul 1x PBS, 200ul buffer AL, & vortex incubate in a thermomixer 15-20 min at 56C, shaking at 600rpm 3. Add 200ul EtOH & vortex 4. With the Qiagen kit, this is the point at which you move the sample into the spin columns. I just pipette the solution out of the 1.5mL tubes and add it to the columns, leaving the paper behind & later discarding it. 5. In the last step of the extraction, I elute twice with 200ul AE buffer, then concentrate the samples down to 30ul, which can be done using concentrator tubes (Montage PCR tubes from Millipore- expensive) or an ethanol precipitation (cheap). This protocol yields 20-40 ng of good quality DNA/ 30ul, depending on how much blood went in at the beginning. I've found that preserving samples in EDTA results in higher quality & yield- for your older samples, you may want to add a bit more proteinase K and incubate longer at 56C. Because the DNA yield varies between samples, you'll want to standardize your concentrations prior to sequencing (we standardize to 20ng in 100ul AE buffer). If you're looking for a cheaper method than the Qiagen kits, I know that Ken does a similar guandine extraction that pellets the DNA rather than using spin columns- I imagine he would send you the protocol if you email him. *****Qiagen kit adaptation***** Qiagen Blood Card Protocol? as adapted by Waits Lab (1/9/08) 1. Turn heat block to 70ºC, check the thermometer. 2. Add a 2-3mm2 piece of blood card to 180ul buffer ATL (be sure to sterilize utensils between cards) and incubate at 55 degrees C for a few hours or overnight. **when Doni did this step overnight, she had a few samples where the liquid had evaporated by morning?for these, she added an equivalent amount of water back to the sample and proceeded with extraction. She says they worked fine** 3. Add 20 µl Proteinase K (20mg/ml) + 200 µl Buffer AL. 4. Vortex immediately, for at least 15 seconds. 5. Incubate at 70ºC for 10 minutes. 6. Add 210 µl 100% Ethanol, vortex. 7. Place sample in a spin column (about 600 µl). 8. Spin at 6000 rpm for 1 minute. 9. Place the filter in a new collection tube. 10. Add 500 µl Buffer AW1 to spin column. Spin at 6000 rpm for 1 minute. 11. Put the filter in a new collection tube and add 500 µl of AW2. Spin at 6000 rpm for 3 minutes. 12. Place the filter in a new 1.5ml microcentrifuge tube with the lid cut off (not provided with the kit). 13. Add 200 µl of Buffer AE and incubate for 2 min at 70ºC. 14. Centrifuge at 8000 rpm for 1 minute. 15. For each sample take the Buffer AE at the bottom of the 1.7ml microcentrifuge tube and pipette it back through the filter. Repeat step 14. (Alternatively, a second aliquot of 200ul of AE can be centrifuged through the filter to produce a total of 400µl of extract as opposed to 200µl ? this is what Doni did with all her samples (both blood cards and tissues) and she ended up needing to do a few extra PCR cycles for the blood card samples). *****Whole Genome Amp and Qiagen MDA kit***** I don't know if you're interested in Whole Genome Amplification (WGA), but we've been working successfully with a protocol to WGA directly from the blood spots. Our reason for doing it was low expected yield anyway, and desire to reduce cost (i.e. not having to do both a DNA extraction, and then a WGA reaction). It's worked very well for us on human blood spots. We use the Qiagen MDA reaction. Let me know if you're interested, and I'll have my tech who worked on the protocol get it to you. *****Qiagen kit***** For human and dog blood we take 20 punches and do SDS/ proteinase K lyses and put through a Qiagen DNA extraction column using their standard methods. The method gives us good amount and quality of DNA. *****Qiagen kits***** I have worked with mammalian blood from FTA/blotting paper. Apparently, there are different extraction procedures according to the type of blotting paper used - however, I couldn't get this information from the sample provider. I used a Qiagen user developed protocol that I found on line (which I don't have to hand but can chase up if you need), and this worked well. But there is also a qiagen method specifically for FTA cards http://www1.qiagen.com/literature/Default.aspx?Term=3Dblood+spot&Language=3DEN&LiteratureType=4%3b8%3b9%3b10&ProductCategory=0 The yield wasn't as high as blood, but better than serum or hair. I increased my amplification cycles in PCR by 10, and then they all seemed to work. I suppose it depends how well optimised you PCR is to weaker samples. The extraction protocols give very specific instructions about how much paper to add, but I was always quite generous. I also had variation in how much blood was on the card, but this didn't make too much difference as all of my samples amplified. *****QIAam DNA Investigator Kit***** I've used the Qiagen QIAamp DNA Investigator Kit, which can extract DNA from just about anything (bubblegum, cigarette butts, cheek swabs, etc.). I noticed that they also have a card specifically for FTA cards. *****Qiagen kits and GeneSolve***** I would be interested in hearing what you find out. I talked to the Whatmann people recently and they now have an FTA elute card that you can "cook" the DNA off of but that won't help for samples on the FTA classic. There is a produce called GeneSolve that is supposed to remove DNA from FTA cards but I haven't tested it. Also in the past I have treated the card punch like a piece of tissue - diced it, put it into lysis buffer, prot-K -> and extracted DNA with a Qiagen spin column. It is not great for recovery but it works when you have a lot of blood on the card. As I work on mammals and you on birds I would expect the success to be higher for you. *****Purgene Kit***** I have done DNA extraction from duck blood from FTA cards many times. I use Puregene chemicals and a Puregene protocol. If you have drops of blood, one drop might yield something like 4-6 ug DNA in total, but that varies according to my experience. The only problem I have sometimes is purity as measured on a nandrop, but that can be solved by additional washing steps in necessary. I don't have a paper where I desribe the method, yet. Please find my own little protocol attached. This is based on a protocol that I can't find anymore, so if there are further issues just let me know. But I've heard from easy to use column spin kits as well. My protocol is cheap but takes a while... Puregene DNA purification Kit-Based on ?DNA purification Protocol for 50ul Human Blood Stains? 1. Cell lysis - Cut FTA card in pieces and place into 1.5ml tube - Add 600 ul Cell Lysis Solution plus 3ul ProtK (20mg/ml) - 55C over night 2. Protein precipitation - Add 200ul Protein Precipitation Solution - Vortex at high speed for 20 sec. - place sample in ice bath for 15 min. - centrifuge at 13k ? 16k x g for 3 min. 3. DNA precipitation - carefully pour supernatant (contains the DNA) into 1.5ml tube containing 600ul 100% Isopropanol. - mix sample gently by inverting 50 times and place in ice bath for 15 min. - centrifuge at 13k ? 16k x g for 5 min. - carefully pour off the supernatant and drain tube on paper. Add 600 ul 70% Ethanol and invert tube several times to wash the DNA pellet. - centrifuge at 13k ? 16k x g for 1 min. - carefully pour off the ethanol and drain tube on paper. Pipet the last drop out and let it air dry for 10-15 min. 4. DNA hydration - Add 20-40ul DNA Hydration Solution. - Rehydrate by incubating for 1h in 65C and/or over night at room temp. *****Purgene Kit 2***** I have done this work very simply: just take a sterile blade and make some scratches on the dried blood spot to obtain some blood "dust" in a recipient. Then, I use a standard kit (we used Puregene kit from Qiagen) following instructions referring to the protocol for DNA extraction from whole blood. We kept the paper disks at -35 in plastic bags. Everything worked well even after months. We got very variable avian blood samples, either for the quantity or the shape of the droplets, from Russian zoos or from the field. I do not expect you will have problems. Look at our paper in press, if you wish, see attached file Barbanera, F et al. In press. Human-mediated introgression of exotic chukar (Alectoris chukar, Galliformes) genes from East Asian into native Mediterranean partridges. Biol. Invasions. *****Sigma Extract-N-Amp kit***** I have been developing FTA card protocols for DNA barcoding in fungi. I have been able to achieve very high rates of PCR success when I used Sigma Extract-N-Amp kits in combination with 2mm discs. For our purposes, we use 25uL of Extraction solution form the Plant kit, incubate according to the instructions (95C for 10min, I think), then add an equal volume of Dilution solution. After that, I dilute 1:9 in water, then use 1-2 uL of the diluted extract in a PCR (10uL total volume) using Invitrogen Platinum Taq (0.1uL) and 2.5mM MgCl2 (final). The Platinum taq is superior to the JumpStart supplied with the kits, but the JumpStart will work. Also, for our PCRs, we found it necessary to increase the MgCl2 to 2.5mM (I think the JumpStart ReadyMix supplied with the Sigma kit is only 1.5mM). Using the Sigma kit we typically get 95+% success. Hopefully you'll find that this works for you! I am in he process of writing these methods up for a publication, so if they do work I'd love to know so that I could mention it in the discussion. *****Nucleospin and "Elute FTA cards***** One of our lab technicians tells me she succesfully extracts DNA from FTA cards using NucleoSpin. Elute with 50 µL and use somewhat more eluted DNA (e.g. 5 µL) for downstream application. On the other hand: did you know there's another type of FTA cards ("Elute") that allows exactly for this? It uses a simple protocol based on elution by water. You end up with something in the range of 30 µL of eluted DNA. Here too, we often use a higher amount as advised as PCR template. However, we work on different life stages of helminth parasites, and I can imagine your blood samples contain a lot more DNA making this change in the procedure perhaps unnecessary. *****Non-kit protocol***** Use 1/16" hole punch; put 3 punches per tube, punch clean matrix between tubes (ie to clean the punch) add 200µl 0.5% SDS, invert twice, incubate 10minutes (room temp) pipette the solution twice, remove all liquid, repeat this wash step add 200µl TE (10mM Tris, 2mM EDTA) invert twice and incubate at room temp for 10 minutes pipette solution twice, remove all liquid, repeat this wash step THEN we made the Whatman "solution 1" which is 0.1N NaOH, 0.3mM EDTA, pH 13.0 *add 35µl solution 1, incubate ONE HOUR at room temp *add 65µl of 0.1M TrisHCl, ph7.0 anf vortex 5 times to mix incubate 10 minutes @ RT vortex 10 times fish out filter papers using pipet tip, squeezing paper against side of tube to remove all liquid Eluate contains gDNA in TE (66mM Tris-HCL, 0.1mM EDTA). We had good recovery from arthropod tissue smears. *****Non-kit protocol 2***** I am currently working on chicken Genomics at the International Livestock research Institute (ILRI) Nairobi Kenya. The scenario you have given below is very much similar to the samples we have in our laboratory. Kindly find attached a useful protocol that has worked quite well for the chicken samples in our lab and should work for any avian samples that are nucleated. The DNA quality is good however, this should be used within a month since degradation occurs and the quality may reduce after this period. For PCR you could play around with quantities ranging from 1ul to more (in a 50ul reaction) depending on the initial quantity of DNA on the FTA. SOP for DNA extraction from FTA cards with whole blood from chicken with high concentration of RBCs 1. Punch five 1.2mm Discs using the Harris punch and place them in 1.5mm Eppendorf tubes. 2. Add 1ml of 100mM (Tris 0.1%SDS) and gently agitate for 30 minutes then discard 3. Add 500ul of 5M Guanidine Thiocynate and agitate for 10 minutes then discard. 4. Add 500ul of triple distilled water and agitate for 10 minutes, do this three times then discard the water. 5. Add 50ul of triple distilled water and place in a water bath at 90 degrees and heat for 10 minutes. 6. Leave to cool at room temperature for 30 minutes. 7. This will generate 50-70ul, Use 1ul for PCR reaction (or optimize accordingly) *****Any kit***** Cutting the disk out and using almost any DNA extraction protocol works great for lizard blood dots or liver smears for us. *****methanol wash?***** Also, one person remembered reading that at least one source recommended a wash with methanol first for the removal of haem *****Plant DNA***** I've played around with extracting plant DNA from Whatman cards. I've attached some previous correspondence I've had about the extraction protocol I used with moderate to poor success. From what I've read, avian DNA is a lot easier to extract, so you might not have to go through all the measures outlined below. Also attached are documents sent to me by Jim Robbins (jim.robbins@whatman.com), who was the scientist at Whatman that I corresponded with at the time about eluting DNA from the cards (of particular interest would be the "FTA elution datasheet", if you haven't run across this yet elsewhere). Finally, the following paper might be useful to you, since it outlines issues particular to avian blood samples: ---------- Forwarded message ---------- Hi John, I had mixed success with getting Whatman paper to work, both with the "regular" protocol (washing a punch and then adding it directly to the PCR) and with the elution methods. My main problem was inhibition- many of the plants I was working with had secondary metabolites which were difficult to wash off. I think the elution protocol I had the most success with was: 1) wash a 3mm punch 2x with 0.5% SDS (the proprietary Whatman buffer didn't improve my results much, so you might as well save a lot of money and use SDS) according to the regular wash protocol 2) wash punch 2x with TE (also according to protocol) 3) follow Whatman elution protocol (attached). The subsequent DNA concentration was higher when I let the punch soak in the 1st (basic) solution for a longer time period (~1h) The success of this method will depend on the species. In the documents attached you'll see that there's been more success with animal tissue. I would suggest playing around with the size of punches and the number of pre-washes (DNA concentration vs. PCR inhibition). Also see some of the suggestions from Jim Robbins in my correspondence with him below. ---------- Forwarded message -------- I'm sorry for my delay in responding to your email. In addition to the washing tips we discussed before, some systems benefit from PCR additives, discussed in an attached document. These apparently tie up some PCR inhibitors and permit amplification. If you do want to elute the DNA from FTA you can do it with controlled damage such as restriction endonucleases, heat or high pH. Our protocol uses high pH but several customers have used Chelex extraction and other methods (attached). In the extreme case you can consider the FTA/DNA complex as if it were a piece of tissue and use chloroform/phenol extraction. If you really want to obtain the DNA in solution then it may be better just to use FTA Elute (www.whatman.com/ftaelute). It offers similar benefits as FTA but uses a hot water treatment to elute the DNA while leaving most PCR inhibitors on the punch. Our work has been overwhelmingly with blood but it would certainly be worth trying with leaves. -----Original Message----- Thank you for your help several months ago in figuring out ways to purify Whatman punches for plants. I confess that I gave up on the washes- they did not work for my species, so I embarked on the "easier" task of isolating DNA from dried leaf tissue. Now, however, I am working on plant samples for a different lab, and do not have dried tissue to fall back on. One species will not amplify at all, regardless of the wash procedure, and the other species only amplifies variably (I think it varies with the punch size - when I punch as small as possible, the amplification works, but such small punches are difficult to produce and work with). PCR reactions from both species are failing due to inhibition (I've been adding positive control DNA to the reactions, which then fail). I have been considering extracting DNA from the paper, as this may allow me to purify it more, and identify what might be inhibiting the reaction. I've been reading up on various protocols, such as high pH and high temperature, and a bunch of variations on washing the punch. It would help me out a great deal if I could understand under which conditions DNA is eluted from the paper- is it mainly high pH, and/or high temperature (and how high?). What kinds of compounds are generally removed with the Whatman buffer wash, and what compounds may be co-eluted at high pH or temperatures? ---------- Quoted text ---------- > Thanks for your call. I think more effective washing of the punches will > give you better PCR from your plant samples. After that it could be useful > to try the smaller punch size as well. > > The isopropanol wash is described in the attached file. The other > alternates we discussed are 0.3M sodium carbonate and 0.5% SDS. In each > case a 5 or 10 minute soak with occasional mixing is recommended. Some labs > have found Proteinase K treatment very useful but, as you said, your samples > are probably not protein-rich. > (Proteinase K method: Add 200 µl of FTA Purification Reagent > containing 60µg/ml Proteinase K to the PCR amplification tube containing the > 1.2 mm FTA sample punch and incubate at 65?C incubator for 30 minutes. > After incubation wash punch 3 times with 200uL FTA Purification Reagent then > 2 times with 200uL of TE-1. No previous washing has to take place before > the Proteinase K treatment. ) > > Adding a washed, sample-containing punch to the positive control PCR tube > can help show whether sample components are inhibiting the amplification. > > I don't think this should be necessary but it is possible to use standard > DNA isolation methods to purify DNA from FTA as if the card were a tissue. > I'll attach an overall publications list so you can look at plant results > and an extraction subset in case eluting the DNA becomes of interest. Our > own recommended elution protocol is an easy high-pH one but it requires > thorough washing of the punch first, which would not help in this case. dacostaj@bu.edu