Amplification of DNA from Whole Blood J. Burckhardt Roche Diagnostic Systems, Business Unit Immunochemistry, F. Hoffmann-La Roche Ltd., CH-4002 Basel, Switzerland

A method is described for the amplification by PCR of human chromosomal DNA sequences from whole blood samples. Various amounts of blood samples, with either EDTA, citrate, or heparin used as the anticoagulant, have been used to determine optimal PCR conditions for each type of sample. Up to 8 0 % (vol/vol) of whole blood sample is tolerated in PCR with TacI polymerase. Amplification from whole blood requires the optimization of salt (K § and Mg §247 according to sample volume and type of anticoagulant used. Pretreatment of fresh blood samples to lyse the leukocytes is required for EDTA-treated blood samples and is beneflcient in PCR with heparin- and citrate-treated blood samples to obtain maximal amplicon amounts. A satisfactory method of pretreating samples is freeze/thawing. In addition, EDTAtreated blood samples require a heat treatment before PCR for maximal amplicon synthesis. It appears that purification of the DNA is not necessary for any of the whole blood samples analyzed by PCR. Results of amplification reactions from unpurifled hepatitis B virus (HBV) sequences of whole sera samples are shown also.

Whole blood, plasma, and sera represent, by far, the most c o m m o n l y used sample types in the diagnostic field. Because PCR inhibitors in blood samples ~ have been described, generally, it is accepted that a careful purification of nucleic acids is required from such samples before PCR analysis can be performed. Classical methods for nucleic acid purification from clinical samples involve reagents and steps like organic solvents, denaturing agents, detergents, centrifugation, and precipitation, ~2'3~ m a n y of which are not compatible with heat-stable DNA polymerases nor desired in future applications with automated PCR systems. Thus, a simple sample preparation m e t h o d allowing DNA analysis by PCR from whole blood would be valuable. Different groups have succeeded in using small amounts of unpurified blood samples as substrate for PCR. Amplification of DNA from 1 I~1 to 2 i~1 of unpurified blood in 100-txl reactions was obtained after a cyclic thermal pretreatm e n t of the samples at 95~ and 55~ ~4~ while it was observed that Tth but not Taq polymerase will amplify DNA from small samples of unpurified and untreated blood. ~s~ The work presented here describes conditions under which chromosomal sequences can be amplified from unpurifled blood using EDTA, heparin, or citrate as anticoagulants. Sample volumes constituted 50% or more of the PCR assay.

MATERIALS AND METHODS PCR Buffer and Solution The lOx concentrated buffers of the L series contain: 50 mM tricine (N-[Tris(hydroxymethyl)-methyl]-glycine), at 25~ (pH 8.8); 15 mM MgC12; 0.5% 3:239-2439

by Cold Spring Harbor Laboratory Press ISSN 1054-9803/94 $5.00

Tween 20; and various KCI concentrations: buffer lOx LO, 0 mM KC1; buffer 10x L1, 100 mM KCI; buffer lOx L2, 200 mM KCI; and so forth up to buffer 10x L15, 1500 mM KC1. The buffers of the LM series have a tenfold higher MgCl2 concentration of 150 mM, but are otherwise identical with the buffers of the L series. The lOx concentrated solution T contains 15 mM MgCl 2 and 0.5% Tween 20.

PCR Setup Reactions were prepared in a laminar flow hood, according to the contamination precautions suggested. (6~ The frozen blood samples were thawed at room temperature. An aliquot was added in 0.5-ml reaction tubes to 4.5-~1 lOx buffer and autoclaved water to obtain a total volume of 45 ~l. The v o l u m e was covered with 2 drops of mineral oil. Sample pretreatment was performed by cycling - 2 0 times between 90~ and 50~ for 1 m i n each in the thermocycler (Landgraf TPS 5/9). A 5-p.1 PCR mix was then added at room temperature containing 0.5 ~l of a 10 mM solution of the dNTP (Pharmacia), 0.5 ~l of the lOx PCR buffer, 1.25-2.5 units of Taq polymerase (usually Super-Taq from P.H. Stehelin, Switzerland), 0.5 ~l of each primer (50 ~M) and water to a final v o l u m e of 5 ~l. Thermocycler conditions for PCR were 30 sec at 93~ then 30 sec at a n n e a l i n g temperature, and 90 sec at 72~ usually for - 4 0 cycles. Reaction tubes were briefly centrifuged after PCR and 7-10 p.l of the supernatant below the oil layer were loaded on 2% TBE agarose gels. The 50 p.1 reactions containing >30% and more of whole blood (vol/vol) were diluted after amplification with 30 ~l of water before centrifugation. Amplicon detection was by e t h i d i u m bromide staining of the DNA in agarose gels. I~.R Methods and Applications

239

Primer Sequences and Annealing Temperatures (AT) Primers were s y n t h e s i z e d in h o u s e o n a Cyclone (Milligen) apparatus. Primers were used after d e b l o c k i n g w i t h o u t further purification. (1) HLA p r i m e r GH26/ 27. (7) AT: 60~ a m p l i c o n size 242 bp. (2) Primers for b l o o d factor IX. (a) JR3 5' AGG ACC GGG CAT TCT AAG CAG TTT A 3'; JR4 5' CAG T I T CAA CTT GTT TCA GAG GGA A 3'; AT: 55~ a m p l i c o n 234 bp. (3) Primer for hepatitis B virus. MD 122 5' CTC TCA ATT TTC TAG GGG GA 3'; MD 123 5' AGC AGC AGG ATG AAG AGG AA 3'; a m p l i c o n 151 bp.

RESULTS The c o n d i t i o n s for DNA PCR from w h o l e blood samples were o p t i m i z e d in two steps (described in sections A a n d B). An extensive p r e t r e a t m e n t of t h e u n p u r i f i e d samples was p e r f o r m e d (see section A) by freeze/thawing once a n d subseq u e n t l y cycling 20 times for l - r a i n each between 90~ a n d 50~ to reach maximal lysis of the w h i t e b l o o d cells before amplification. This sample p r e t r e a t m e n t e n a b l e d the d e t e r m i n a t i o n of o p t i m a l salt c o n c e n t r a t i o n s , assay sensitivity, a n d m a x i m a l w h o l e b l o o d sample volumes tolerated in PCR assays. Subsequently, fresh w h o l e b l o o d samples were used to d e t e r m i n e the m i n i m a l conditions of sample p r e t r e a t m e n t for PCR of leukocytes (section B).

A1. Optimal KCI and MgCI 2 Concentrations in PCR with Whole Blood Samples It has b e e n s h o w n t h a t Taq p o l y m e r a s e has a KCI o p t i m u m of 50 mM. (9) Blood has a K + a n d Na § c o n c e n t r a t i o n of 124 mM. (1~ Thus, t h e i n f l u e n c e o n PCR of DQA1 of various m o n o v a l e n t salt conc e n t r a t i o n s was c o m p a r e d u s i n g as substrate either 20 n g h u m a n DNA or 20% w h o l e b l o o d w i t h EDTA as a n t i c o a g u l a n t (data n o t s h o w n ) . A broad salt o p t i m u m was f o u n d w i t h best salt c o n c e n t r a t i o n s in the PCR buffer r a n g i n g from 50 mM to > 1 0 0 mM KC1 for purified DNA a n d from 0 mM to 90 mM KC1 for w h o l e b l o o d samples. KC1 c o n c e n t r a t i o n s < 5 0 mM in the buffer yield specific products o n l y w i t h w h o l e b l o o d samples b u t n o t w i t h purified DNA as substrate. A c o n c e n t r a t i o n of 150 mM KC1 allows s o m e DNA synthesis

240

PCR Methods and Applications

w i t h purified DNA, but not w i t h w h o l e b l o o d samples. T h e n the o p t i m a l salt c o n c e n t r a t i o n was d e t e r m i n e d for PCR w i t h S-S0% of pretreated w h o l e blood (Table 1). All three types of a n t i c o a g u l a n t s in the b l o o d allow specific a m p l i c o n synthesis for the tested volumes. The PCR buffer w i t h the usual KC1 c o n c e n t r a t i o n of 50 mM enables specific DNA synthesis in the presence of up to 30% EDTA-treated b l o o d and up to 50% heparin-treated blood, while h i g h KCI (and h i g h MgCI2) c o n c e n t r a t i o n s are required for the amplification from citrate-treated blood samples. The m i n i m u m KC1 r e q u i r e m e n t was d e t e r m i n e d by replacing the PCR buffer w i t h a s o l u t i o n T c o n t a i n i n g o n l y MgC12 a n d Tween 20, but no buffering substance or KC1. This solution allows specific and efficient a m p l i c o n synthesis from large v o l u m e s of EDTA- a n d heparin-treated blood, but not of citratetreated blood. O p t i m i z a t i o n of MgC12 concentrations for u n p u r i f i e d citrate-treated blood (20% a n d S0% sample volumes) gave best values w i t h MgC12 r a n g i n g b e t w e e n 4.5-15 mM, in the presence of 50-100 mM KCI (data n o t shown). Efficient DQA1 synthesis is f o u n d w i t h 1.5 mM MgC12 in the PCR buffer in reactions w i t h all v o l u m e s of h e p a r i n blood samples. In a m p l i f i c a t i o n s w i t h large EDTAtreated blood samples, the m i n i m a l

MgCI 2 c o n c e n t r a t i o n appears to be - 1 mM above the EDTA c o n c e n t r a t i o n , w h i c h ranges in u n d i l u t e d EDTA-treated blood b e t w e e n 3.5 a n d 4.0 mM.

A2. Sensitivity of PCR Assays with Whole Blood Samples H u m a n EDTA-treated b l o o d was m i x e d in various ratios w i t h sheep EDTAtreated blood to analyze the sensitivity of the PCR assay w i t h 20% u n p u r i f i e d blood sample (Fig. 1). It was established previously t h a t the p r i m e r pair GH 26/27 is specific for h u m a n DQA1 a n d gives n o PCR p r o d u c t w i t h the h o m o l o g o u s g e n e of sheep. The results s h o w that 10 nl of h u m a n blood (with - 5 0 leukocytes) in a 10-1:L1 sheep blood s a m p l e is sufficient to yield a positive signal in 40 amplification cycles, even w h e n s t a i n i n g amplicons by e t h i d i u m b r o m i d e in agarose gels, an u n s e n s i t i v e DNA d e t e c t i o n procedure.

A3. Confirmatory Experiments W h o l e b l o o d samples (20% a n d 40% vol/ vol) from 19 d o n o r s were assayed in 50p.l a m p l i f i c a t i o n reactions: ten samples w i t h EDTA-treated b l o o d (Fig. 2), five with h e p a r i n - t r e a t e d b l o o d (Fig. 3A), a n d four w i t h citrate-treated b l o o d (data n o t shown). No s p e c i m e n s s h o w e d detectable i n h i b i t i o n of PCR.

TABLE 1 O p t i m i z a t i o n of Salt C o n c e n t r a t i o n s in PCR Assays of DQA1 w i t h 5 - 5 0 % Pretreated EDTA-, Citrate-, or Heparin-treated Blood

Heparin-treated blood 0 mM KC1 (T) 50mMKCl(LS) EDTA-treated blood 0 mM KC1 (T) 50mMKCl(LS) Citrate-treated blood 0 mM KC1 (T) 0 mM KCI, 15 mM Mg* * (LOM) 50 mM KCI (L5) 50 mM KCI, 15 mM Mg* § (LSM) 100 mM KC1 (LIO) 1OO mM KC1, 15 mM Mg + * (LIOM) 150 mM KC1, 15 mM Mg* * (L1SM)

S

Sample percentage (vol/vol) in PCR 10 20 30 40

++

+ + + +

+ + + +

+ + ++

+ + ++

+ + + +

-/U ++

+/U + +

+ + + +

+ + ++

+ + -

+ + -

+ +rid + + . + + +

+ +AJ + +

. -AJ + + -/U + + + + + +

.

. -/U + + +/U + + + + + +

. -FU + + +/U . + + + +

. +rid + + . . + + +

50

.

+ -

Amplification in 40 cycles of DQA1 from whole blood samples in varying KCI and MgC12 concentrations (for PCR buffer compositions, see Materials and Methods). ( - ) No amplicon visible on agarose gel; ( + ) low amount of amplicon visible; ( + + ) large or very large amounts of amplicon visible; (U) unspecific DNA synthesis.

C o n f i r m a t i o n of the results o b t a i n e d for the DQA1 a m p l i c o n was s o u g h t with the primers J3/4 specific for a sequence of the b l o o d factor IX. (8) Various sample v o l u m e s of h e p a r i n - t r e a t e d b l o o d from five different d o n o r s were tested, all giving the expected a m p l i c o n of 234 bp (data n o t shown). In addition, PCR for DQA1 with heparin-treated b l o o d was p e r f o r m e d in reactions where Taq p o l y m e r a s e was substituted with Pfu, rTth, or Vent, all three enzymes giving positive results (data n o t shown).

A4. Amplification of Large Volumes of Blood Samples The m a x i m a l sample v o l u m e of citrateor EDTA-treated w h o l e b l o o d w h i c h gave visible a m p l i c o n yields was 60% (data n o t shown). Heparin-treated b l o o d on the other h a n d is better tolerated by Taq polymerase: a m p l i c o n s are even f o r m e d (with decreasing efficiency) in reactions c o n t a i n i n g up to 82% b l o o d (Fig. 3B). A further increase in w h o l e b l o o d sample v o l u m e analyzed in a single amplification reaction was o b t a i n e d by increasing the sample and reaction volumes at the same time. PCR e x p e r i m e n t s

FIGURE 2 DQA1 was amplified from 10 ~1 and 20 ~1, respectively, of pretreated EDTA-treated blood from 10 different blood donors in L0 buffer for 40 cycles. Lanes with even number: 20% blood samples per PCR. Lanes with uneven numbers: 40% blood samples per PCR. (Lanes I and 2) Donor 1; (lanes 3 and 4) donor 2; and so on. Blood from donor 7 was assayed in duplicate (lanes 13-16).

were p e r f o r m e d using 200 ill of whole heparin-treated b l o o d in reaction volumes of 500 ~1. All reagents were used in the same p r o p o r t i o n as in the usual 50-1J.l reaction. Cycling c o n d i t i o n s were exp a n d e d from 3 m i n to 10 min. Comparable a m p l i c o n yields were o b t a i n e d with samples of different donors analyzed in large or small reaction v o l u m e s (Fig. 3A and data not shown).

Alternatively, storing heparin- a n d citrate-treated b l o o d samples for - 5 days at r o o m t e m p e r a t u r e before PCR, gives h i g h a m p l i c o n yields w i t h o u t a n y sam-

B. Amplification with Fresh Blood Samples

FIGURE 1 Sensitivity of PCR assay with 10 ~1 unpurified blood. Human EDTA-treated blood was mixed with sheep EDTA-treated blood in various concentrations, pretreated, and amplified with 40 cycles in L2 buffer. The following amounts of human blood were present per reaction: (lane 1) 10 I~1;(lane 2) 1 i~1; (lane 3) 100 nl; (lane 4) 10 nl; (lane 5) 1 nl; (lane 6) 0.1 nl; (lane 7) 0.01 nl. (Lane 8) PCR with 10 IJ.1sheep blood only. (M) Marker.

The results m e n t i o n e d so far were obtained with blood samples aliquoted 2 days after d o n a t i o n and stored frozen before p e r f o r m i n g PCR after sample pretreatment. The following e x p e r i m e n t was designed to analyze m i n i m a l pret r e a t m e n t c o n d i t i o n s for fresh, w h o l e b l o o d samples. Samples stored at r o o m t e m p e r a t u r e for 2 hr (heparin- and citrate-treated blood) or 24 hr (EDTA-treated blood) after d o n a t i o n were used w i t h o u t any pret r e a t m e n t , after freeze/thawing in dry ice, or after freeze/thawing and cycling 5 times between 90~ and 50~ (Table 2). It appears that heparin- and citratetreated b l o o d samples can be used directly for PCR, but that p r e t r e a t m e n t by freezing is giving o p t i m a l a m p l i c o n synthesis. M i n i m a l sample p r e t r e a t m e n t for EDTA-treated b l o o d consists of a freeze/ t h a w step. Further p r e t r e a t m e n t by from 5 up to 15 a d d i t i o n a l heat cycles is necessary for o p t i m a l a m p l i c o n yields with EDTA-treated blood.

FIGURE 3 Amplification of DQA1 from large heparin-treated blood samples. (A) 100 IJ.land 200 ~1, respectively, of pretreated heparintreated blood from 5 different donors was used as substrate in PCR assays of 500-~1 reaction volume, with solution T as PCR buffer. Amplification was for 40 cycles of 10 min each, with 3 min 20 sec for each step (denaturation at 92~ annealing at 60~ and elongation at 72~ The supernatant (7 i~1) was loaded after a 1-min centrifugation on a 2% agarose gel as in the other experiments. Lanes with even numbers: 200 IJ.lblood (or 40% voll vol) present in the amplification reaction; lanes with uneven numbers: 100 i~l blood per reaction. (Lanes 1 and 2) Blood from donor 1; (lanes 3 and 4) donor 2; and so on. (B) Amplification reactions with very large fractions (40-82% vol/vol) of pretreated blood were analyzed. Reactions of 50 i~1were set up with 10 i~1, 20 IJ.1, 30 i~1, 35 IJ.l, and 41 ~1 of sample, respectively, for 40 cycles, with solution T replacing PCR buffer (lanes 1-5, respectively).

PCR Methods and Applications

24 !

is released very easily in PCR buffer w i t h Tween 20.

D e t e r m i n a t i o n o f M i n i m a l Sample P r e t r e a t m e n t Necessary for Amplification of DQA1 from Fresh Blood Samples of Two Donors, A a n d B

TABLE 2

Sample pretreatment before PCR no pretreatment ( - ) , freezing ( + ) EDTA-treated blood A,B A,B A,B Heparin- or citrate-treated blood A,B A,B A,B

S denaturation cycles (90-S0~

Amplicon synthesis

+ +

§

+ + ++

+ +

+

+ + + + +

Amplification for 43 cycles in buffer L3 (EDTA- and heparin-treated blood) or L10M (citratetreated blood) of DQA1 from 20~ (vol/vol) fresh whole blood without any pretreatment or pretreated as indicated. Freezing was done for 30 min at - 70~ All results for blood samples of donor A and B were identical. Symbols for the results are as in Table 1.

pie p r e t r e a t m e n t , w h i l e aged, EDTAtreated b l o o d samples still require freeze/ t h a w i n g a n d from 5 to 15 h e a t cycles for o p t i m a l a m p l i f i c a t i o n (data n o t shown).

C. Amplification of Hepatitis B Virus (HBV) from Whole Serum Preliminary experiments showed that DQA1 can be a m p l i f i e d in reactions w i t h 10% to 40% s e r u m spiked w i t h 20 ng h u m a n DNA a n d buffer L5 c o n t a i n i n g 50 mM KCI after s e r u m p r e t r e a t m e n t (freeze/thawing a n d 20 cycles of pretreatm e n t at 90~176 n o t shown). The sensitivity of t h e n e w p r o c e d u r e was further a n a l y z e d for PCR of HBV DNA: serum w i t h a titer of 15 • 106 HBV gen o m e s per microliter (serum a n d HBV titer d e t e r m i n a t i o n was p r o v i d e d by Dr. U. H e n n e s a n d B. Wolf) was diluted to various c o n c e n t r a t i o n s w i t h a HBV-negative control serum. Aliquots of 10 ~1 of sera were pretreated as described a n d amplified in 50-1~1 reactions w i t h HBVspecific primers (Table 3). Samples cont a i n i n g ~ 1S00 viral g e n o m e s gave visible a m o u n t s of the expected a m p l i c o n of 151 bp after 40 PCR cycles.

DISCUSSION DNA PCR by Taq p o l y m e r a s e of sequences from pretreated, w h o l e b l o o d samples treated w i t h h e p a r i n , EDTA, or citrate as a n t i c o a g u l a n t is possible, even w i t h samples c o n t r i b u t i n g 50% or m o r e to the reaction v o l u m e . According to the present study o n l y two variables h a v e to

242

PCR Methods and Applications

be taken into a c c o u n t w h e n a m p l i f y i n g DNA from w h o l e blood: (1) Accessibilty of target sequence to the PCR reagents. (2) Adaptation of salt c o n c e n t r a t i o n to the e n z y m e requirements. A further factor, i n a c t i v a t i o n of inhibitor(s) of the enz y m a t i c a m p l i f i c a t i o n reaction, seems n o t to be necessary (see below).

Accessibility Cellular or viral DNA, present in vivo in a protected form in cells or virus, is n o t readily accessible to all PCR reagents. This barrier is overcome by lysing the cells or virus. The o p t i m a l lysis treatm e n t differs d e p e n d i n g o n the type of a n t i c o a g u l a n t used (Table 2). It could be s h o w n that c h r o m o s o m a l DNA from cells in heparin- or citrate-treated blood

Amplification of DNA Sequence from HBV from W h o l e Serum

Salt concentration

The Na + a n d K ++ c o n c e n t r a t i o n of w h o l e b l o o d a m o u n t s to an average of 124 mM, the MgCl 2 c o n c e n t r a t i o n averages 5.1 mM. The respective values for plasma are 145 mM a n d 1.6 raM. r176 The c o n t r i b u t i o n of m o n o v a l e n t salt for t h e a n t i c o a g u l a n t h e p a r i n is b e l o w 1 mM, 10 mM for EDTA as a t r i p o t a s s i u m salt, a n d 36 mM for t r i s o d i u m citrateJ TM Because of the salt c o n c e n t r a t i o n present in PCR w i t h large b l o o d samples, it appears unnecessary or even i n h i b i t o r y to add m o r e p o t a s s i u m to PCR assays w i t h h e p a r i n - or EDTA-treated b l o o d samples, w h i l e t h e r e q u i r e m e n t of p o t a s s i u m for citrate blood samples is very h i g h (overview in Table 4).

Blood as Inhibitor of PCR The p u b l i s h e d observations o n i n h i b i tion of PCR using w h o l e b l o o d (type of a n t i c o a g u l a n t often n o t reported) a n d our o w n results n e e d n o t be contradictory. 11-3'12-14) Citrate-treated blood, w h i c h is t h e m o s t c o m m o n l y used b l o o d t r e a t m e n t in diagnostics, requires an unusually h i g h salt buffer for PCR. Leukocytes in EDTA-treated b l o o d o n the o t h e r h a n d are difficult to lyse. It seems likely t h a t PCR w i t h EDTA- or citrate-treated blood could have given the negative results for these reasons rather t h a n for t h e presence of s o m e i n h i b i t o r y substance. Heparin-treated w h o l e blood, w h i l e appearing to be the best suited type of w h o l e b l o o d sample for DNA PCR, is unlikely to have b e e n used because of the w e l l - k n o w n i n h i b i t o r y action of h e p a r i n

TABLE 3

Number of HBV genomes per PCR assay

TABLE 4

Amplicon synthesis

1

1.S • 106

+ +

2 3 4 S 6 7

1.S • lO s 1.S • 104 1SO0 150 15 negative control serum

++ ++ +

Amplification of HBV from 20% (vol/vol) pretreated sera was in buffer LS for 40 cycles. Symbols used are as in Table 1.

M o n o v a l e n t Salt Necessary in PCR Buffer for O p t i m a l A m p l i f i c a t i o n of DNA from Various A m o u n t s of W h o l e Blood Samples Sample volume (%) Whole blood

10

20

30

40

50

EDTA-treated Heparin-treated Citrate-treated

50 70 100

25 50 100

0 20 80

0 0 50

0 0 50

Optimal K ~ concentrations (mM) for PCR with whole blood samples containing different anticoagulants.

in PCR w i t h purified DNA or RNA substrate. (is) H e p a r i n i n h i b i t s restriction enzymes a n d p o l y m e r a s e s due to b i n d i n g with h i g h e r affinity of these e n z y m e s to h e p a r i n t h a n to t h e DNA. (16~ It is a surprising result t h a t h e p a r i n is n o t interfering in w h o l e b l o o d PCR in contrast with PCR w i t h partially purified DNA or RNA w i t h copurified h e p a r i n w h e r e inh i b i t i o n b y h e p a r i n c o u l d be o v e r c o m e by digestion of t h e s a m p l e u s i n g heparinase. (17> It is a s s u m e d t h a t a l b u m i n (~8'19~ or other b l o o d c o m p o n e n t s a b s e n t in purified samples are able to sequester heparin d u r i n g PCR w i t h w h o l e b l o o d samples. Also, it is u n l i k e l y t h a t the results s h o w n in this work are caused by an artifact. C o n t a m i n a t i o n , the m a i n cause of false-positive results in PCR, is n o t a crucial aspect for e x p e r i m e n t s d e s i g n e d to overcome an i n h i b i t o r y action of a sample. In a n y case the c o n t a m i n a t i o n level in the e x p e r i m e n t s s h o w n is less t h a n the n u m b e r of DNA copies present in about 50 leukocytes (Fig. 1). In addition, the fact t h a t efficient a m p l i c o n s y n t h e s i s in PCR w i t h large b l o o d samples is possible w i t h different p o l y m e r a s e s a n d w i t h different target sequences argue against the p r o b a b i l i t y t h a t the described observations w o u l d be valid in special cases only. Finally, a n d m o s t important in this regard, are two very recent p u b l i c a t i o n s in w h i c h PCR is described w i t h specific types of large w h o l e blood samples. (2~ The a m p l i c o n s s y n t h e s i z e d in the presence of w h o l e s e r u m (and sometimes plasma; data n o t s h o w n ) are often unstable, an observation n o t m a d e for w h o l e b l o o d PCR: a m p l i f i c a t i o n products of sera c a n n o t be stored for 24 hr at r o o m t e m p e r a t u r e unless stabilized by adding EDTA (final c o n c e n t r a t i o n , 20 mM) or p r o t e i n a s e K (50 p.g/reaction). In addition, samples s o m e t i m e s b e c o m e viscous, a fact t h a t seems n o t to interfere w i t h DNA amplification, but m a d e amplicon d e t e c t i o n difficult. D i l u t i o n of the samples after PCR w i t h 30 ~1 water and/or proteinase K d i g e s t i o n for 30 rain at 56~ h e l p e d to reduce this p r o b l e m . W h o l e b l o o d samples n e v e r t u r n e d viscous n o r h a d u n s t a b l e a m p l i c o n s , even in reactions w i t h large b l o o d v o l u m e s where n e a r l y all reaction v o l u m e was t a k e n up b y precipitated b l o o d c o m p o nents. O t h e r applications of the n e w procedure: PCR from very large b l o o d samples

(200 p.l) in 500-p.1 reactions (this work), from dried b l o o d samples, (2z) or w h o l e serum samples (23,24) (and this work) n o w have been described at least to some e x t e n t for PCR w i t h u n p u r i f i e d substrate. In conclusion, h e p a r i n i z e d w h o l e b l o o d samples appear to be the best suited type of w h o l e b l o o d for DNA PCR w i t h Taq polymerase: (1) Heparintreated blood can be added directly to a PCR assay. (2) Taq tolerates up to 82% h e p a r i n b l o o d in a PCR assay. (3) Amplicon synthesis by Taq is efficient w i t h h e p a r i n - t r e a t e d blood. (4) No decrease in specificity or efficiency of a m p l i c o n synthesis was f o u n d w h i c h was caused by different sample volumes; t h a t is, low sensitivity of Taq to variation of salt or o t h e r reaction c o m p o n e n t s in PCR w i t h h e p a r i n - t r e a t e d blood.

12.

13.

14.

15.

16. REFERENCES 1. Higuchi, R. 1989. Simple and rapid preparation of samples for PCR. In PCR technology (ed. H.A. Erlich), pp. 31-38. Stockton Press. 2. Loparev, V.N., M.A. Cartas, C.E. Monken, A. Velpandi, and A. Srinivasan. 1991. An efficient and simple method of DNA extraction from whole blood and cell lines to identify infectious agents. I. Virol. Methods 34:105-112. 3. Walsh, P.S., D.A. Metzger, and R. Higuchi. 1991. Chelex 100 as a medium for a simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 10: 506-513. 4. Mercier, B., C. Gaucher, O. Feugeas, and C. Mazurier. 1990. Direct PCR from whole blood, without DNA extraction. Nucleic Acids Res. 18: 5908. 5. Panacchio, M. and A. Lew. 1991. PCR in the presence of 8% (v/v) blood. Nucleic Acids Res. 19: 1151. 6. Kwok, S. and R. Higuchi. 1989. Precautions for PCR. Nature 339: 237-238. 7. Erlich, H.A. and T.L. Bugawan. 1989. HLA class II gene polymorphism: DNA typing, evolution, and relationship to disease susceptibility. In PCR technology (ed. H.A. Erlich), pp. 193-208. Stockton Press. 8. Reiss, J., U. Neufeldt, K. Wieland, and B. Zoll. 1990. Diagnosis of haemophilia B using the polyrnerase chain reaction. Blut 60: 31-36. 9. Gelfand, D. 1989. Taq DNA polymerase. In PCR technology (ed. H.A. Erlich), pp. 17-22. Stockton Press. 10. In Documenta Geigy 1970. Scientific tables: Blood; inorganic substances (ed. K. Diem and C. Lentner), edition 7, pp. 561568, published by J.R. Geigy S.A., Basle. 11. NCCLS Document H1-A3: Evacuated

17.

18. 19.

20.

21.

22.

23.

24.

tubes for blood specimen collection, third edition, 1991. Vol. 11/9, pp. 1-17. Beutler, E., T. Gelbart, and W. Kuhl. 1990. Interference of heparin with the polymerase chain reaction. BioTechniques 9" 166. Holodny, M., S. Kim, D. Katzenstein, M. Konrad, E. Groves, and T.C. Merigan. 1991. Inhibition of human immunodeficiency virus gene amplification by heparin. ]. Clin. Microbiol. 29" 676-679. Wang, J.-T., T.-H. Wang, J.-C. Sheu, S.-M. Lin, J.-T. Lin, and D.-S. Chen. 1992. Effects of anticoagulants and storage of the blood samples on efficacy of the polymerase chain reaction assay for hepatitis C virus. J. Clin. Microbiol. 30: 750-753. Chen, J., L.A. Herzenberg, and L.A. Herzenberg. 1990. Heparin inhibits EcoRI endonuclease cleavage of DNA at certain EcoRI sites. Nucleic Acids Res. 18" 32553258. Bickle, T.A., V. Pirrotta, and R. lmber. 1977. A simple, general procedure for purifying restriction endonucleases. Nucleic Acids Res. 4" 2561-2572. Imai, H., O. Yamada, S. Morita, S. Suehiro, and T. Kurimura. 1992. Detection of HIV-1 RNA in heparanized plasma of HIV-1 seropositive individuals. J. Virol. Methods 36" 181-184. Hagelberg, E. and B. Sykes. 1989. Ancient bone DNA amplified. Nature 342: 485. Higuchi, R. 1992. Dr. Russ problem corner. In Ancient DNA newsletter (ed. R.K. Wayne and A. Cooper), vol. l, pp. 6-8. Institute of Zoology, The Zoological Societv of London, London. Panaccio, M., M. Georgesz, and A.M. Lew. 1993. FoLT PCR: A simple PCR protocol for amplifying directly from whole blood. BioTechniques 14- 238-243. McCusker, J., M.T. Dawson, D. Noone, F. Gannon, and T. Smith. 1992. Improved method for direct PCR amplification from whole blood. Nucleic Acids Res. 20" 6747. Nelson, P.V., W.F. Carey, and C.P. Morris. 1990. Gene amplification directly from Guthrie blood spots. Lancet 336: 14511452. Frickhofen, N. and N.S. Young. 1991. A rapid method of sample preparation for the detection of DNA viruses in human serum by polymerase chain reaction. J. Virol. Methods 35: 65-72. Cheyrou, A., C. Guyomarc'h, P. Jasserand, and P. Blouin. 1991. Improved detection of HBV DNA by PCR after microwave treatment of serum. Nucleic Acids Res. 19: 4006.

Received September 20. 1993; accepted in revised form December 16, 1993.

I~CR Methods and Applications

243