FTA^® paper is a commercial product (Whatman at ) consisting of filter paper impregnated with a proprietary mix of chemicals which serve to lyse cells, to prevent growth of bacteria, and to protect the DNA in the sample.

The basic premise of purifying DNA using FTA^® paper is simple: biological samples are applied to the FTA^® paper and air-dried. A small disc of the FTA^® paper is then removed, and washed to remove any non-DNA material (the DNA remains entangled within the paper). Analysis can subsequently be performed on the DNA whilst still attached to the paper, or the DNA can be eluted prior to use.

Examples of washing and processing procedures, and of the utility of DNA processed in this manner, are presented in this manuscript.

For vertebrates, the common sample of choice is generally blood or blood clots, as the cells are often still living and the DNA is usually of high quality. After processing, the resulting concentration of DNA per unit area of FTA^® paper is also usually fairly constant within each species. This reliability simplifies many procedures by removing the need for DNA quantitation prior to analysis. Although more variable, cheek wipes and saliva deposited on FTA^® are satisfactory samples, although such samples include some DNA that has been damaged by apoptotic nucleases as well as bacterial DNA. None of this may be important for procedures such as PCR as long as some of the DNA of the animal is intact; however it may cause amplification-variations due to the highly variable amounts of DNA present.

The reagents on the FTA^® paper are designed to kill most pathogens, inhibit saprophytes during drying or bouts of high humidity, and have strong buffering and free-radical-trap properties to deal with atmospheric pollutants.

Room temperature transport in folders or envelopes (by hand or mail) has been common for years. The papers protect DNA within the samples for some years at ambient conditions. The main variable is expected to be the storage atmosphere quality, particularly the content of acid gases and free-radical-generating pollutants, although FTA^® paper can protect against such conditions [5]. Definite storage times can therefore only be stated with reference to the load of these agents in the atmosphere. However forensic samples have been stored on these papers for more than a decade in ordinary office filing cabinets whilst still remaining amplifiable. From the basic chemistry of DNA damage it is reasonable to expect that storage times may be considerably enhanced if atmospheric circulation is deliberately inhibited.

From basic principles of DNA chemistry, sample integrity would be expected to vary with the cleanliness of the storage atmosphere and the amounts of preservatives on the papers. In a western-style city in a Mediterranean climate (South Australia) avian blood samples were placed onto FTA^® paper, and were stored in an ordinary filing box in a laboratory cupboard (to restrict atmospheric circulation) in the dark for up to 44 months. Air conditioning was intermittent, and therefore room temperature ranged from 18°C to 42°C. Discs were removed from these samples, processed by method #4 (see below), and used in a PCR reaction to amplify ILRC loci [4]. All samples amplified successfully, (figure 1) demonstrating that the DNA has not undergone significant degradation during storage. Thus, once dried, blood and tissue samples placed onto FTA^® paper require no specialised storage or transport conditions, and the DNA within the samples remains intact for at least 4 years stored in a card file at room temperature.

In most methods a disc of appropriate size is first punched from the tissue-containing paper; 1 mm diameter for PCR of mammalian DNA from a blood or clot, and up to 5 mm in diameter for a RFLP study. The paper has to be completely dry in order to punch a disc cleanly, and the disc is then washed and dried. For most PCR applications the DNA is amplified in situ; for RFLP or cloning of DNA, the DNA is digested while still attached to the small washed paper discs. Cross contamination due to paper carryover by the punch is not a problem as paper fibres do not adhere to the sample punch, but 'clean' discs may be punched between samples if required.

The examples below involve sample processing by simple washing carried out in eppendorf tubes, multiwell plates or similar. Wash fluid is dropped on and the washes are thrown away while the DNA remains on the paper until either removed or PCR amplified in-situ. Fine tipped needles or pipettes are best used for aspirating wash solutions as with coarse tipped devices the small discs may also be aspirated. Experience has shown that often neither the volumes or the times quoted below are critical and should thus be viewed as suggestions. During the washing, only gentle rocking is needed and violent agitation is undesirable. Indeed, with the 1 mm discs rocking may be dispensed with if extra changes of solution are added to the protocol, as the disturbance from the extra solution changes is sufficient agitation. The discs are usually dried in an ordinary lab heating block or oven for 15~20 min.

Processing method #1 – A 1 mm disc of FTA^® paper was placed in 200 μl 1% SDS, (sodium dodecyl sulphate), 2 mm EDTA (pH 8.0) for 10 minutes. The SDS was removed with three changes of 200 μl Tris-EDTA buffer (TE) buffer (5 minutes each wash) and the TE was removed with a few seconds wash with water followed by 200 μl ethanol or isopropanol.

Processing method #2 – A 1 mm disc of FTA^® was washed twice with 500 μl 10 mm NaOH (each wash for 15 minutes) and then washed with 500 μl TE:water (1:5) containing 1 mg/ml BSA for 15 minutes.

Processing method #3 – A 1 mm disc was washed twice (5 minutes each) with 200 μl liquid phenol (Commercial available water-saturated phenol). The disc was washed twice in 250 μl 80% isopropanol containing 5% tris (free-base) and then dried at 55°C.

Examples of the end use of DNA processed by these methods can be seen in the amplification of ILRC PCR products (ILRCs are multi-locus markers, which may be considered as similar to microsatellites) from mammalian blood (figure 2a,2b,2c).

In all methods the disc can be placed directly in the PCR mix after processing, and PCR amplified in situ.

The above methods are older procedures that have been reported to work well with microbial [6] and mammalian DNA with direct PCR.

Method #4 is a method that can cope with the very high levels of DNA found in blood from vertebrates with nucleated erythrocytes (e.g. birds, fish and reptiles). Variations of this method, as required dependant on the source of the sample and the final use of the DNA, are described in Table 1 (see additional file 1).

This procedure involves a more thorough deproteinisation than the previous procedures because the DNA is to be removed from the disc or digested in-situ.

Discs of FTA^® paper containing avian blood samples were washed with 1 ml (100 mM Tris (free base), 0.1% SDS), agitating gently for 30 minutes. The discs were then washed in 500 μl 5 M guanidine thiocyanate or one of the commercial DNA purifying solutions such as DNAzol^© (Life Technologies at ). Finally the discs were washed 3 times with 500 μl water and once with 500 μl 95% ethanol. The DNA-loaded discs were now ready for procedures such as restriction digestion or DNA elution.

Bird blood contains too much DNA for even the smallest punched discs to be used directly in PCR reactions. For example six blood samples from Australian pelicans (Pelecanus conspicilattus) were processed by method #4 and three of the 1 mm discs were placed into 50 μl water and heated to 90°C for 10 minutes to release much of the DNA. Eluant (1 μl) was used in the subsequent PCR. The three remaining discs were used directly in the PCR as is usual practice with mammalian samples. PCR amplification of the CHD gene was performed, and all six samples then analysed by gel electrophoresis (figure 3a). The samples with the 1 μl eluant as template (that is to say only one fiftieth or less of the total DNA on a disc) amplified well while direct amplification on the FTA^® punches (with the full load of DNA) was poor or failed resulting in smearing on the gel. This is the opposite of the case with mammalian blood (non-nucleated erythrocytes) and was apparently due to the gross excess of DNA introduced into the PCR reaction from the nucleated red blood cells.

FTA^® punches from 24 pelican blood samples were processed by method #4 and extracted for 10 minutes with 50 μl water at 90°C. Eluted DNA (1 μl) was then used as template in a subsequent DNA amplification to identify the sex of the birds by amplification of the CHD gene and subsequent restriction digestion.

All but one sample yielded a clear result (figure 3b) at the first amplification (this sample was subsequently successfully amplified), with the total time required for sex identification (processing, PCR, restriction of product and analytical gel) being less than 8 hours.

With samples containing nucleated erythrocytes, heating to 90°C in water was suitable for detaching DNA from the FTA^® paper for PCR, although once released from the paper the DNA may not be stable (either at 4°C or -20°C) in water for long periods (> 2 weeks). Once the sample has been well washed with deproteinising agents such as guanidine thiocyanate, DNA is readily digestable and may be released by restriction endonucleases in-situ for RFLP analysis or cloning. The release of digested DNA from the paper was examined using a cocktail of restriction enzymes, to completely digest any DNA present. After digestion the DNA fragments should be stripped from the discs using centrifugation, as even very low molecular weight DNA diffuses too slowly to leave the paper completely in short time periods.

A stripping exercise was performed to illustrate these issues: A 1 mm disc of FTA^® paper containing chicken blood was washed as normal (method #4). After digestion with a cocktail of restriction enzymes in a 50 μl volume (to completely digest the entangled DNA) the bottom of the reaction-tube was punctured with the tip of a scalpel blade, the tube placed into a larger tube, and centrifuged in a standard bench centrifuge at 13 000 rpm for 30 seconds to strip all solution from the paper. TE (50 μl) was then added to the paper (twice) and the tube re-centrifuged. The paper was subsequently transferred into a fresh tube, 50 μl water added and heated to 90°C for 10 minutes, to remove any large DNA fragments still attached to the paper. Finally, the residual paper was sealed into the well of an agarose gel. All DNA samples were analysed by agarose gel electrophoresis (figure 4). From the gel tracks it can be seen that most DNA was recovered by a simple spin elution (Lane 1), with little extra DNA being yielded by the subsequent spin elutions with TE (Lanes 2a,2b). Heating to 90°C released some extra DNA, particularly larger fragments (Lane 3) such as restriction-site-deficient macrosatellite DNA which had remained entangled in the paper, whilst even after this procedure some DNA was retained on the paper (Lane 4) again, presumably, macrosatellite DNA.

This apparent size-selection may have an application in the enrichment of DNA for particular restriction resistant DNA fragments.

Blood samples from chicken, cow, crested pigeon (Geophaps lophotes) and sleepy lizard (Tiliqua rugiosa) were spotted onto FTA^® paper as normal. The blood samples from the chicken and cow contained anticoagulant (heparin). Small tissue fragments (generally muscle) approximately 2 to 4 mm in diameter from mallee fowl (Leipoa ocellata), frog (Rana sp.), yabbie (an Australian fresh water crustacean, Cherax tenuimanus), abalone (Haliotis laevigata) and blue swimmer crab (Portunus pelagicus) were placed onto FTA^® paper and manually crushed into the weave of the paper using a microscope slide to shield the thumb from the sample. Two sizes of disc (1 mm and 5 mm diameter) were first processed following method #4, and the discs digested overnight with 10 units of AluI (nominally an amount capable of digesting 10 μg of DNA per hour.) The centrifugally-stripped DNA was analysed by agarose gel electrophoresis (figure 5a). As expected, observable amounts of DNA were only observed from the 1 mm discs from species that have nucleated red blood cells (birds, lizard, fish) although only small amounts of DNA were observed from frogs. Observable amounts of DNA were eluted from 5 mm punches from all species, nucleated or not, from blood samples and other tissues. The 5 mm punches of blood samples from some species with nucleated red blood resulted in incompletely digested DNA, presumably due to the large amounts of DNA present on the disc. King George Whiting (Sillaginodes punctata) and tuna (Thunnus maccoyii) blood samples were also processed similarly. Observable amounts of DNA were eluted from 1 mm discs, and large amounts of incompletely digested DNA were eluted from 5 mm discs.

Thus, as a general principle, restricting DNA from FTA^® to be used in cloning should be preceded by observing a trial restriction of a 1 mm disc so as to determine the amount of DNA present in the particular organism's tissue sample.

Blood samples were collected from pelicans, and plasma removed. The residual blood cells were then allowed to dry at room temperature for several days under non-sterile conditions before being resuspended in water and placed onto FTA^® paper. Using method #4 initial attempts at amplification failed, indicating that significant degradation of the sample had occurred. However, by omitting the deproteinisation wash (and increasing the number of cycles in the PCR), PCR amplification of the CHD gene was possible (figure 7a) suggesting that residual protein was serving to hold DNA fragments onto the paper during the processing procedure.

Fractions of some of the original blood samples were phenol:chloroform extracted, and the DNA examined by gel electrophoresis (figure 7b). The DNA in all samples was almost totally degraded. However processing of these blood samples on FTA^® paper, without deproteinisation, followed by PCR did result in amplification of the CHD gene (and sex identification) in 4 out of the 5 samples. Therefore, collection of moderately decayed samples, while not reliable, may be a worthwhile procedure.

PCR for the amplification of the avian CHD gene was performed as described [3]. PCR for amplification of avian and mammalian DNA repeat sequences (ILRCs) was as previously described [4]. Gels are standard 1% agarose TBE gels.

Digestions were performed in 50 μl volumes, in buffers appropriate to the restriction enzyme as supplied by the manufacturer. The processed FTA^® discs were placed directly into the enzyme-containing buffer and incubated overnight (generally at 37°C, but as appropriate for the restriction enzyme being used).