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A simplified rapid method for restriction fragment length polymorphism analysis after bone marrow transplantation
ELSEVIER
JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 168 (1994) 183-185
A simplified rapid method for restriction fragment length polymorphism analysis after bone marrow transplantation Thomas Schreiner *, Markus Wiesneth, Birgit Maccari, Beate Sawodny, Heike Prochnow-Calzia, Bernhard Kubanek Red Cross Blood Center and Department of Transfusion Medicine, University of Ulm, Ulm, Germany
(Received5 August 1993, accepted 21 September 1993)
Abstract
A rapid and simple protocol for restriction fragment length polymorphism (RFLP) analysis using digoxigenin labeled DNA probes is presented. The method described gives consistently good results even with minute cell numbers. The method has been used to evaluate chimerism after bone marrow transplantation but could also find other applications. Key words: Restriction fragment length polymorphism; Chimerism analysis; Bone marrow transplantation; (Method)
1. M e t h o d
3 X 10 6 nucleated ceils were used for a standard D N A purification procedure or 105 cells for a small scale purification [latter values are given in brackets in the following text]. After lysis of the red blood cells from an E D T A blood sample by 0.5% Triton X-100, the nucleated cells were resuspended in 1 ml [100/zl] of proteinase K buffer (50 mm Tris-HC1 p H 7.4, 5 mm CaCI2, 150 mm NaCI) in 2 ml [0.5 ml] E p p e n d o r f tubes. 10/~1 [1 /~1] of proteinase K (100 m g / m l ) and 25 txl [2.5 /~1] of 20% SDS were added and the solution
* Corresponding author. At: DRK Blutspendezentrale, Helmholtzstrasse 10, D-89081 Ulm, Germany. Tel.: 0049/731/150-197; Fax: 0049/731/150-175.
incubated for 30 min at 65°C with constant shaking in a thermo mixer (Eppendorf). The D N A was immediately precipitated with isopropanol, washed and redissolved in 500 /zl [50 tzl] of T E (10 mm Tris-HCl pH 7.5, 1 mm EDTA). 0.6 /~g genomic D N A was digested with 4 U of AvalI and separated on a 0.8% horizontal agarose gel. In order to compress the low molecular weight bands the gel was placed at a 3 ° angle in the electrophoresis buffer. The D N A was blotted and U V cross-linked to a Nytran N nylon membrane (Schleicher & Schiill) with the posi blot system (Stratagene). The multilocus D N A probe hMF1 (Tynan and Hoar, 1989) was cloned into the SmaI site of p G E M - 3 Z f (Promega). For PCR labeling 100 ng plasmid D N A was incubated with 200 ~mol dNTPs, 20 /.~mol D I G - d U T P , 50 pmol T7 and
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(93)E0253-E
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T. Schreiner et al. / Journal of lmmunological Methods 168 (1994) 183-185
SP6 primer and 2 U Taq polymerase (Amersham) in Taq polymerase buffer (Amersham). The DNA was amplified in 40 cycles (95°C, 1 min; 57°, 1 min; 72°, 2 min). 5 /~1 of the PCR solution were used per ml of hybridization solution without further purification. Hybridization for 2 h and detection of the bands with NBT were performed with the digoxigenin kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions.
~R ~R ,D ,D
2. Results and discussion
The average yield of high molecular weight DNA from 3 x 106 ceils was 20-25/zg and from 105 cells 0.6-0.8 /zg. The OD ratio 260:280 nm was constantly above 1.7, indicating a clean DNA preparation. Furthermore the DNA was completely dissolved in TE after a few minutes which is also an indicator of pure DNA. DNA prepared by the salting-out procedure (Miller et al., 1988) or extracted with phenol/chloroform (Sambrook et al., 1989) was not superior to DNA prepared according to our method (data not shown). In order to obtain optimal results it is important to pay attention to two points: the cell numbers and buffer volumes selected were shown to give the best results in terms of DNA purification. Higher cell concentrations lead to a poorer quality of DNA whereas our technique can be scaled down to 2.5 × 104 cells. Proteinase K digestion in the presence of Ca 2÷ ions and NaC1 gives purer undegraded DNA than does digestion with EDTA. Mixing the samples thoroughly throughout the period of incubation disperses clumps of cells and is an essential determinant of the purity of the final DNA preparation. Chimerism analyses after bone marrow transplantation using the present method are shown in Fig. 1. The main advantage of the present RFLP technique is that the complete purification of DNA is performed in one tube in less than 1 h. Furthermore, the DNA never passes through a pipette tip thereby preventing degradation of the nucleic acid by shearing and the technique does not require the handling of toxic substances. Direct labeling of DNA with digoxigenin and plas-
Fig. 1. Southern blot of AvalI digested genomic D N A from five bone marrow-donor/recipient pairs. Patients 1 - 4 reveal complete chimerism post bone marrow transplantation. Patient 5 shows complete chimerism of the BM-cells (B) on day 40 post BMT. O n day 52 the peripheral P M C (P) shows mixed chimerism and the M N C (M) a complete recipient-specific pattern indicating graft rejection. D = donor, R = recipient before BMT, P = polymorphonuclear cells of the patient after BMT, M = mononuclear cells of the patient after BMT, B = bone marrow of the patient after BMT, D • = donor specific band, R • = recipient specific band.
mid specific primers in the PCR gives high yields of full length probe. The sensitivity of detection is increased several fold compared to random labeling. Even 0.3 /zg genomic DNA per lane give clear results and the buffer gradient compresses and sharpens the low molecular weight bands. Because to its sensitivity and the availability of low cell numbers the present method was successfully used in more than 300 chimerism analyses very early after bone marrow transplantation and during severe aplasia after buffy coat therapy (Hertenstein et al., 1992). HLA-DR typing with probe pRTV1 (Bidwell and Jarrold, 1986) was performed in addition to detection of the single copy gene nm23 (Steeg et al., 1988) from human leukocytes. The speed and simplicity of the present protocol renders it applicable to all kinds of RFLP analyses.
T. Schreiner et al. /Journal of lmrnunological Methods 168 (1994) 183-185
3. References Bidwell, J. and Jarrold, E.A. (1986) HLA-DR allogenotyping using exon-specific cDNA probes and application of rapid minigel methods. Mol. Immunol. 23, 1111-1116. Hertenstein, B., Bunjes, D., Wiesneth, M., Heinze, B., Novotny, J., Heimpel, H. and Arnold, R. (1992) Immunophenotypical and haematological changes in patients receiving interferon a and donor buffy coat transfusions for treatment of CML relapse after allogeneic bone marrow transplantation. Ann. Hematol. 65, A68. Miller, S., Dykes, D. and Polesky, H. (1988) A simple salting
185
out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tynan, K. and Hoar, D. (1989) Primate evolution of a human chromosome 1 hypervariable repetitive element. J. Mol. Evol. 28, 212. Steeg, P., Bevilacqua, G., Kopper, L., Thorgeirsson, U.P., Talmadge, J.E., Liotta, L.A. and Sobel, M.E. (1988) Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst. 80, 200-204.
JOURNALOF IMMUNOLOGICAL METHODS Journal of ImmunologicalMethods 168 (1994) 183-185
A simplified rapid method for restriction fragment length polymorphism analysis after bone marrow transplantation Thomas Schreiner *, Markus Wiesneth, Birgit Maccari, Beate Sawodny, Heike Prochnow-Calzia, Bernhard Kubanek Red Cross Blood Center and Department of Transfusion Medicine, University of Ulm, Ulm, Germany
(Received5 August 1993, accepted 21 September 1993)
Abstract
A rapid and simple protocol for restriction fragment length polymorphism (RFLP) analysis using digoxigenin labeled DNA probes is presented. The method described gives consistently good results even with minute cell numbers. The method has been used to evaluate chimerism after bone marrow transplantation but could also find other applications. Key words: Restriction fragment length polymorphism; Chimerism analysis; Bone marrow transplantation; (Method)
1. M e t h o d
3 X 10 6 nucleated ceils were used for a standard D N A purification procedure or 105 cells for a small scale purification [latter values are given in brackets in the following text]. After lysis of the red blood cells from an E D T A blood sample by 0.5% Triton X-100, the nucleated cells were resuspended in 1 ml [100/zl] of proteinase K buffer (50 mm Tris-HC1 p H 7.4, 5 mm CaCI2, 150 mm NaCI) in 2 ml [0.5 ml] E p p e n d o r f tubes. 10/~1 [1 /~1] of proteinase K (100 m g / m l ) and 25 txl [2.5 /~1] of 20% SDS were added and the solution
* Corresponding author. At: DRK Blutspendezentrale, Helmholtzstrasse 10, D-89081 Ulm, Germany. Tel.: 0049/731/150-197; Fax: 0049/731/150-175.
incubated for 30 min at 65°C with constant shaking in a thermo mixer (Eppendorf). The D N A was immediately precipitated with isopropanol, washed and redissolved in 500 /zl [50 tzl] of T E (10 mm Tris-HCl pH 7.5, 1 mm EDTA). 0.6 /~g genomic D N A was digested with 4 U of AvalI and separated on a 0.8% horizontal agarose gel. In order to compress the low molecular weight bands the gel was placed at a 3 ° angle in the electrophoresis buffer. The D N A was blotted and U V cross-linked to a Nytran N nylon membrane (Schleicher & Schiill) with the posi blot system (Stratagene). The multilocus D N A probe hMF1 (Tynan and Hoar, 1989) was cloned into the SmaI site of p G E M - 3 Z f (Promega). For PCR labeling 100 ng plasmid D N A was incubated with 200 ~mol dNTPs, 20 /.~mol D I G - d U T P , 50 pmol T7 and
0022-1759/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0022-1759(93)E0253-E
184
T. Schreiner et al. / Journal of lmmunological Methods 168 (1994) 183-185
SP6 primer and 2 U Taq polymerase (Amersham) in Taq polymerase buffer (Amersham). The DNA was amplified in 40 cycles (95°C, 1 min; 57°, 1 min; 72°, 2 min). 5 /~1 of the PCR solution were used per ml of hybridization solution without further purification. Hybridization for 2 h and detection of the bands with NBT were performed with the digoxigenin kit (Boehringer Mannheim, Germany) according to the manufacturer's instructions.
~R ~R ,D ,D
2. Results and discussion
The average yield of high molecular weight DNA from 3 x 106 ceils was 20-25/zg and from 105 cells 0.6-0.8 /zg. The OD ratio 260:280 nm was constantly above 1.7, indicating a clean DNA preparation. Furthermore the DNA was completely dissolved in TE after a few minutes which is also an indicator of pure DNA. DNA prepared by the salting-out procedure (Miller et al., 1988) or extracted with phenol/chloroform (Sambrook et al., 1989) was not superior to DNA prepared according to our method (data not shown). In order to obtain optimal results it is important to pay attention to two points: the cell numbers and buffer volumes selected were shown to give the best results in terms of DNA purification. Higher cell concentrations lead to a poorer quality of DNA whereas our technique can be scaled down to 2.5 × 104 cells. Proteinase K digestion in the presence of Ca 2÷ ions and NaC1 gives purer undegraded DNA than does digestion with EDTA. Mixing the samples thoroughly throughout the period of incubation disperses clumps of cells and is an essential determinant of the purity of the final DNA preparation. Chimerism analyses after bone marrow transplantation using the present method are shown in Fig. 1. The main advantage of the present RFLP technique is that the complete purification of DNA is performed in one tube in less than 1 h. Furthermore, the DNA never passes through a pipette tip thereby preventing degradation of the nucleic acid by shearing and the technique does not require the handling of toxic substances. Direct labeling of DNA with digoxigenin and plas-
Fig. 1. Southern blot of AvalI digested genomic D N A from five bone marrow-donor/recipient pairs. Patients 1 - 4 reveal complete chimerism post bone marrow transplantation. Patient 5 shows complete chimerism of the BM-cells (B) on day 40 post BMT. O n day 52 the peripheral P M C (P) shows mixed chimerism and the M N C (M) a complete recipient-specific pattern indicating graft rejection. D = donor, R = recipient before BMT, P = polymorphonuclear cells of the patient after BMT, M = mononuclear cells of the patient after BMT, B = bone marrow of the patient after BMT, D • = donor specific band, R • = recipient specific band.
mid specific primers in the PCR gives high yields of full length probe. The sensitivity of detection is increased several fold compared to random labeling. Even 0.3 /zg genomic DNA per lane give clear results and the buffer gradient compresses and sharpens the low molecular weight bands. Because to its sensitivity and the availability of low cell numbers the present method was successfully used in more than 300 chimerism analyses very early after bone marrow transplantation and during severe aplasia after buffy coat therapy (Hertenstein et al., 1992). HLA-DR typing with probe pRTV1 (Bidwell and Jarrold, 1986) was performed in addition to detection of the single copy gene nm23 (Steeg et al., 1988) from human leukocytes. The speed and simplicity of the present protocol renders it applicable to all kinds of RFLP analyses.
T. Schreiner et al. /Journal of lmrnunological Methods 168 (1994) 183-185
3. References Bidwell, J. and Jarrold, E.A. (1986) HLA-DR allogenotyping using exon-specific cDNA probes and application of rapid minigel methods. Mol. Immunol. 23, 1111-1116. Hertenstein, B., Bunjes, D., Wiesneth, M., Heinze, B., Novotny, J., Heimpel, H. and Arnold, R. (1992) Immunophenotypical and haematological changes in patients receiving interferon a and donor buffy coat transfusions for treatment of CML relapse after allogeneic bone marrow transplantation. Ann. Hematol. 65, A68. Miller, S., Dykes, D. and Polesky, H. (1988) A simple salting
185
out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Tynan, K. and Hoar, D. (1989) Primate evolution of a human chromosome 1 hypervariable repetitive element. J. Mol. Evol. 28, 212. Steeg, P., Bevilacqua, G., Kopper, L., Thorgeirsson, U.P., Talmadge, J.E., Liotta, L.A. and Sobel, M.E. (1988) Evidence for a novel gene associated with low tumor metastatic potential. J. Natl. Cancer Inst. 80, 200-204.