k.) 1991 Oxford University Press
4294 Nucleic Acids Research, Vol. 19, No. 15
Slide PCR: DNA amplification from cell samples microscopic glass slides
on
Eric P.H.Yap and James O'D.McGee* University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK Submitted June 13, 1991 The polymerase chain reaction (PCR) has been used to amplify sequences in polypropylene tubes from a wide range of template material. We have developed a method for amplifying DNA from cultured cells and smears directly on microscopic glass slides ('slide PCR'). Cells were either allowed to adhere as a monolayer or smeared onto silanised microscope slides (1), then fixed in methanol/acetic acid 3:1 (v/v), Carnoy's solution, absolute alcohol or 4% buffered paraformaldehyde for 5-15 min. These slides were rinsed in distilled water, dried, and then either used fresh or stored at -20°C until use. A 20x38 mm area on the slide was circumscribed using an immunohistochemistry barrier pen (Dako). 30 pl of PCR mix comprising 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2,200 AM dNTP's, 100 nM primers, 0.01% (w/v) gelatin (Sigma), 0.2% (w/v) bovine serum albumin (BSA) and 2.5 U/100 Asl Taq polymerase (Cetus) was pipetted onto the slide. A 22 x40 mm glass cover slip was then placed over the rectangular area of the slide and its margins were sealed with mineral oil (Sigma). The slides, placed on the metal block of a thermal cycler (e.g. Koch-Light) such that there was maximum contact between the block and sample, were subjected to 30-40 rounds of amplification with parameters identical to those required for PCR in propylene tubes. Denaturation temperatures for later cycles could be lowered for short products (2). After the reaction, the cooled slides were then gently dipped into chloroform to remove most of the mineral oil, but without dislodging the cover slip. One corner of the rectangular cover slip was then carefully raised with a sharp pair of forceps and the PCR mix, which collected as a meniscus at the opposite corner, was retrieved with a pipette. Typically, at least 25 A1 could be collected; this solution was run on agarose gels or reamplified with nested primers as for standard tube PCR reactions. 0.1-1 % BSA is necessary for slide PCR. In its absence, amplification did not occur with cell samples on slides or with extracted DNA pipetted directly and dried on plain slides. Precoating slides with BSA allowed amplification though at lower efficiency. Gelatin (at least 0.001 %) was also required for amplifying targets of about 1 kb, but did not affect the yield of smaller products. Slide PCR was successful with the different sample preparation and fixation methods discussed. The mechanism of slide PCR seems to be that DNA is partially eluted from the cells during the initial denaturation, and amplification takes place in the aqueous phase above the cells and slide. No significant amount of DNA was eluted after the *
To whom correspondence should be addressed
initial cycle and there was sufficient DNA left after 30 cycles to be easily detected by in-situ hybridisation (1) with a digoxigenin-labelled total human genomic DNA probe; routine cell staining revealed little loss of morphological detail. Slide PCR provides an alternative for analysis of cell samples that are aleady on slides without the need for scraping them off (3), and also minimises manipulation and risk of contamination. This technique may also be used where only the original microscopic samples are available and need to be preserved for patient follow-up (e.g., cervical and haematological smears).
REFERENCES 1. 2. 3. 4. 5. 6.
Bums,J. et al. (1988) J. Clin. Pathol. 40, 858-864. Yap,E.P.H. and McGee,J.O'D. (1991) Nucl. Acids Res. 19, 1713. Hanson,C.A. et al. (1990) Am. J. Pathol. 137, 1-6. Shibata,D.K. et al. (1988) J. Exp. Med. 167, 225-230. Saiki,R.K. et al. (1986) Nature (London) 324, 163-166. Thein,S.L. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 3924-3928.
Figure 1. Slide PCR amplification of 110 bp sequence of HPV16 (4) of (a) CaSki (300-copy) and (b) SiHa (single-copy) cells. Negative controls are slides of (c) MCF7 (HPV-negative) cells, (d) blank slide and (e) CaSki cells in absence of BSA. Tube PCR of CaSki DNA (f) is performed with the same master mix and cycling parameters (2).
El -
Figure 2. PCR of 110 bp (5) (b, c) and 900 bp (6) (d, e) sequences of (3-globin from CaSki cells on slides (b, d) and in tubes (c, e).
4294 Nucleic Acids Research, Vol. 19, No. 15
Slide PCR: DNA amplification from cell samples microscopic glass slides
on
Eric P.H.Yap and James O'D.McGee* University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK Submitted June 13, 1991 The polymerase chain reaction (PCR) has been used to amplify sequences in polypropylene tubes from a wide range of template material. We have developed a method for amplifying DNA from cultured cells and smears directly on microscopic glass slides ('slide PCR'). Cells were either allowed to adhere as a monolayer or smeared onto silanised microscope slides (1), then fixed in methanol/acetic acid 3:1 (v/v), Carnoy's solution, absolute alcohol or 4% buffered paraformaldehyde for 5-15 min. These slides were rinsed in distilled water, dried, and then either used fresh or stored at -20°C until use. A 20x38 mm area on the slide was circumscribed using an immunohistochemistry barrier pen (Dako). 30 pl of PCR mix comprising 10 mM Tris pH 8.3, 50 mM KCl, 1.5 mM MgCl2,200 AM dNTP's, 100 nM primers, 0.01% (w/v) gelatin (Sigma), 0.2% (w/v) bovine serum albumin (BSA) and 2.5 U/100 Asl Taq polymerase (Cetus) was pipetted onto the slide. A 22 x40 mm glass cover slip was then placed over the rectangular area of the slide and its margins were sealed with mineral oil (Sigma). The slides, placed on the metal block of a thermal cycler (e.g. Koch-Light) such that there was maximum contact between the block and sample, were subjected to 30-40 rounds of amplification with parameters identical to those required for PCR in propylene tubes. Denaturation temperatures for later cycles could be lowered for short products (2). After the reaction, the cooled slides were then gently dipped into chloroform to remove most of the mineral oil, but without dislodging the cover slip. One corner of the rectangular cover slip was then carefully raised with a sharp pair of forceps and the PCR mix, which collected as a meniscus at the opposite corner, was retrieved with a pipette. Typically, at least 25 A1 could be collected; this solution was run on agarose gels or reamplified with nested primers as for standard tube PCR reactions. 0.1-1 % BSA is necessary for slide PCR. In its absence, amplification did not occur with cell samples on slides or with extracted DNA pipetted directly and dried on plain slides. Precoating slides with BSA allowed amplification though at lower efficiency. Gelatin (at least 0.001 %) was also required for amplifying targets of about 1 kb, but did not affect the yield of smaller products. Slide PCR was successful with the different sample preparation and fixation methods discussed. The mechanism of slide PCR seems to be that DNA is partially eluted from the cells during the initial denaturation, and amplification takes place in the aqueous phase above the cells and slide. No significant amount of DNA was eluted after the *
To whom correspondence should be addressed
initial cycle and there was sufficient DNA left after 30 cycles to be easily detected by in-situ hybridisation (1) with a digoxigenin-labelled total human genomic DNA probe; routine cell staining revealed little loss of morphological detail. Slide PCR provides an alternative for analysis of cell samples that are aleady on slides without the need for scraping them off (3), and also minimises manipulation and risk of contamination. This technique may also be used where only the original microscopic samples are available and need to be preserved for patient follow-up (e.g., cervical and haematological smears).
REFERENCES 1. 2. 3. 4. 5. 6.
Bums,J. et al. (1988) J. Clin. Pathol. 40, 858-864. Yap,E.P.H. and McGee,J.O'D. (1991) Nucl. Acids Res. 19, 1713. Hanson,C.A. et al. (1990) Am. J. Pathol. 137, 1-6. Shibata,D.K. et al. (1988) J. Exp. Med. 167, 225-230. Saiki,R.K. et al. (1986) Nature (London) 324, 163-166. Thein,S.L. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 3924-3928.
Figure 1. Slide PCR amplification of 110 bp sequence of HPV16 (4) of (a) CaSki (300-copy) and (b) SiHa (single-copy) cells. Negative controls are slides of (c) MCF7 (HPV-negative) cells, (d) blank slide and (e) CaSki cells in absence of BSA. Tube PCR of CaSki DNA (f) is performed with the same master mix and cycling parameters (2).
El -
Figure 2. PCR of 110 bp (5) (b, c) and 900 bp (6) (d, e) sequences of (3-globin from CaSki cells on slides (b, d) and in tubes (c, e).
