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Genetic alterations in brain tumors identified by RAPD analysis
Gene 206 (1998) 45–48
Genetic alterations in brain tumors identified by RAPD analysis Dil-Afroze a,b, Anjan Misra b,1, Irshad M. Sulaiman a, Subrata Sinha b, Chitra Sarkar c, Ashok K. Mahapatra d, Seyed E. Hasnain a,* a Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India b Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India c Department of Pathology, All India Institute of Medical Sciences, New Delhi, India d Department of Neurosurgery, All India Institute of Medical Sciences, New Delhi, India Received 15 May 1997; received in revised form 23 September 1997; accepted 23 September 1997; Received by J. Wild
Abstract We report the utility of random amplified polymorphic DNA (RAPD) analysis for identifying subtle genomic alterations in ` meningiomas and gliomas by comparing the DNA band profile of tumor vis-a-vis its constitutional counterpart. Twenty out of the 29 decanucleotide GC-rich random primers utilized for the RAPD analysis of meningiomas revealed alteration(s) in the tumor genome. In gliomas, changes were detected by 16 of the 18 primers. While all the seven meningioma samples exihibited alterations in tumor DNA, changes were evident in 21 of the 24 glioma cases. These alterations in tumor DNA included the loss of a normal band, appearance of a new band and amplification of a pre-existing band. Many primers detected more than one alterations in a given tumor. Our approach, which covers the range from 0.4 to 2 kb, besides detecting a significant number of changes in a spectrum of brain tumors, complements existing DNA fingerprinting methods, such as microsatellite mapping ( less than 0.4 kb) and Southern blotting (over 2 kb), for detecting genetic alterations in tumors. © 1997 Elsevier Science B.V. Keywords: Meningioma; Glioma; PCR; Genomic alterations
1. Introduction A number of molecular and cytogenetic methods have been used to identify and characterize genetic alterations in tumors. Of these, the locus non-specific methods of DNA fingerprinting have the potential of identifying novel changes in tumor DNA. Southern hybridization with multi-locus fingerprinting probes has been able to identify changes in malignancies of the gastrointestinal tract, breast, ovary, urinary bladder, meninges, glia and in melanomas ( Thein et al., 1987; de Jong et al., 1988; Chen et al., 1991; Agurell et al., 1992; Matsumura and Tarin, 1992; Joshi et al., 1996). Microsatellite mapping * Corresponding author. Tel: +91 11 6103799/6189622, ext. 301; Fax: +91 11 616-2125; e-mail: [email protected] 1 The first two authors contributed equally to this work. Abbreviations: bp, base pair(s); EtdBr, ethidium bromide; kb, kilobase(s); PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA. 0378-1119/97/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 5 79 - 9
( Vogelstein et al., 1989; Armour and Jeffreys, 1992; Boland, 1996) with polymorphic single locus probes requires a battery of probes depending on the extent of the chromosomal region to be characterized. The advantages of random amplified polymorphic DNA (RAPD) analysis lie in the technique being locus non-specific and easy identification of the regions of amplification, deletion or rearrangement without prior information about the loci permitting simultaneous screening of multiple regions of the genome ( Welsh and McClelland, 1990; Williams et al., 1990; del Tufo and Tingey, 1994; Benter et al., 1995; Matioli and de Brito, 1995; Cheah and Paigen, 1996). Furthermore, RAPD screening can be performed on a small amount of tumor DNA, and it is relatively easier to clone the altered fragments for further analyses. In the present study, we describe the use of RAPD marker-based DNA fingerprinting for comparing the amplification profiles of tumor DNA in meningiomas and gliomas with their normal leucocyte.
46
Dil-Afroze et al. / Gene 206 (1998) 45–48
2. Experimental and discussion 2.1. PCR amplification with RAPD primers Surgically resected tumor samples were collected from the Neurosurgery Operation Theatre of All India Institute of Medical Sciences, New Delhi, India. Tissue was snap-frozen immediately after surgery and frozen sections cut from the entire tumor tissue. Regions of the tumor having more than 80% neoplastic cells were used for preparing DNA. Leucocytes of same patients were the source of the corresponding normal DNA. 10-mer oligodeoxynucleotide primers of random but definite sequences of 50–80% GC content were procured from GENOSYS, USA. Amplification was carried out in 25-ml reaction volume with varying MgCl concen2 trations (1.5–6 mM ) and annealing temperatures (22–48°C ) using Taq DNA polymerase. The PCR products were resolved by electrophoresis on a 1.5% agarose gel and visualised by ethidium bromide ( EtdBr) staining. Paired samples ( leucocyte and tumor DNA) from seven meningioma and 24 glioma patients were analysed. We scored only those bands as altered where comparison could be made on the basis of intensities of the preceding and succeeding bands in the paired sample. 2.2. RAPD analyses reveal differences between the tumor and the constitutive DNAs Twenty-nine primers ( Table 1) were utilized for screening the meningioma genome for alterations, while 18 primers were used for scoring changes in the gliomas ( Table 2). Of these, nine produced amplification products that were monomorphic (Fig. 1, lanes 1–8) and the remaining 20 detected changes (Fig. 2 lanes 1–8) when the meningioma tumor RAPD profile was compared to that of its normal leucocyte DNA. Two of the 18 primers used for screening gliomas generated a monomorphic profile ( Fig. 1, lanes 9–16), while 16 revealed changes ( Fig. 2, lanes 9–16). A multiband profile was observed for all the primers with the total number of bands per primer varying from 2 to 14 for meningiomas ( Table 1) and from 8 to 22 for gliomas ( Table 2). The size of these bands ranged from 400 bp to 2 kb. With a given primer, all seven meningiomas and 21 out of 24 glioma samples exhibited alteration(s). The alterations exhibited in tumor DNA included the loss of a normal band (Fig. 2, lanes 2, 12 and 16), appearance of a new band (Fig. 2, lanes 4, 6, 8 and 14) and amplification of a preexisting band ( Fig. 2, lanes 8 and 10). Many primers detected more than one such alteration in a given tumor ( Fig. 2, lane 8). RAPD amplified fragments are best resolved between 400 bp and 2 kb, whereas the other fingerprinting methods are best for fragments either larger than 2 kb (Southern blotting) or less than 400 bp (microsatellite
Table 1 Primers utilized for RAPD analysis to score alteration(s) in meningiomas Primer
Sequence (5∞–3∞)
Number of amplification productsa
Polymorphism statusb
2 3 5 7 10 13 14 16 18 26 27 28 32 34 35 36 37 39 41 42 47 48 50 52 54 71 77
CAATGCGTCT AGGATACGTG CGGATAACTG TCCGACGTAT CCATTTACGC TACACTAGCG CATAGCCCTT AGTGAATGCG TTTACGGTGG AAGATAGCGG CCTATCCGTT GATTGCGTTC GTCCTACTCG GTCCTTAGCG GTCCTCAACG CTACTACCGC GAGTCACTCG CGTCGTTACC TGCGCGATCG GCAGGATACG AACGTACGCG CGATGAGCCC CATCCCGAAC CAGGGTCGAC ATCTCCCGGG GCACCCGACG GCACGCCGGA
10 7 11 6 9 10 7 13 10 8 5 6 8 7 5 7 2 7 9 7 9 6 7 14 5 6 12
+ + + + + + − + + + + − − + + + − − + − + + − + + − +
aMean of total number of bands generated with the primer. b‘−’ and ‘+’ denote monomorphic and altered band patterns, respectively, when RAPD profiles for tumor DNA was compared with its constitutive counterpart.
mapping). RAPD analysis covers the range in between and could complement these two DNA fingerprinting techniques. In studies exploiting a similar approach, changes in colorectal cancer were demonstrated by Arbitrarily Primed PCR using primers varying in length from 20 to 28 bases (Peinado et al., 1992; Ionov et al., 1993; McClelland and Welsh, 1994). These changes were demonstrated by autoradiography after PAGE. Our study using 10-mer primers extends the application of this approach to brain tumors besides identifying alterations simply at the level of EtdBr-stained agarose gels. Another possible utility of the procedure is for the development of a simple system for scoring the overall extent of genomic alterations in tumors. Genomic instability is now taken as a measure of the extent of expansion and contraction of repeats at a microsatellite locus (Boland, 1996). Altered RAPD fragments are the manifestation of an unstable genome, and the significance of these in terms of the behavior of a tumor needs to be elucidated. Characterization of such altered fragments would constitute the first step in our understand-
Dil-Afroze et al. / Gene 206 (1998) 45–48
47
Table 2 Primers utilized for RAPD analysis to score alteration(s) in gliomas Primer
Sequence
Number of amplification productsa
Polymorphism statusb
9 24 43 39 59 61 64 70 73 76 82 85 87 88 90 95 96 101
AGAAGCGATG GTTAGTGGCA CTGCGATACC CGCTGTTACC TGCAGCACCG GCCCCTCTTG GGACCGCTAG CGCAGACCTC CCATGGCGCC GCACGGAGGG GCCCCATGCG CACCTGCCGC CGCCAGGAGC CGCGTCAGCC TGGTCGGCGC GCGAATTCCG CGGAATTCGC GGCTGCAGCG
15 9 8 16 18 15 16 12 12 22 17 8 13 11 6 9 11 14
+ + + − + + + + + + + + + − + + + +
aMean of total number of bands generated with the primer. b‘−’ and ‘+’ denotes monomorphic and altered band patterns respectively, when RAPD profiles for tumor DNA was compared with its constitutive counterpart.
Fig. 1. Random amplified polymorphic DNA profile generated by 10-mer primers for paired samples of meningioma and glioma exhibits a monomorphic band pattern. A representative primer-template combination profile generated for a single pair of meningioma with four different primers namely P28, P42, P51, P71 ( lanes 1–8) and glioma samples from two patients with 2 different primers P88, P49 ( lanes 9, 10 and 13, 14 with one pair and lanes 11, 12 and 15, 16 with another pair of sample) are shown. Leucocyte and tumor DNA are indicated by N and T, respectively.
ing of the molecular mechanism leading to the onset of tumorigenesis. Further, based on the information on sequence analysis of these altered fragments, this study could be extended to address the possibility of generating RAPD-derived SCAR markers (Gutierrez-Adan et al., 1997) to detect specific alteration(s) in these forms of brain tumors.
Fig. 2. Polymorphism detected by RAPD primers within the tumor genome. The polymorphic regions within the genomes of meningiomas and gliomas when compared to their normal profile appeared in the form of loss of a normal band ( lanes 2, 12 and 16), appearance of a new band ( lanes 4, 6, 8 and 14) or amplification of a preexisting band ( lanes 8 and 10). A representative RAPD profile using a single pair of meningioma ( lanes 1–8) and glioma ( lanes 9–16) samples with 4 different primers is shown.
3. Conclusions (1) Our study demonstrates that RAPD analysis with 10-mer primers of a random but defined sequence can be used to score subtle alterations in genomes of a large proportion of gliomas and meningiomas. (2) The RAPD marker-based procedure for detecting genetic alterations during tumorigenesis utilizes only nanogram quantities of tissue to begin with and requires simple EtdBr staining for scoring these genomic alterations in brain tumors. (3) RAPD amplified fragments varying from 400 bp to 2 kb covering the range in between Southern blotting ( larger than 2 kb) or microsatellite mapping ( less than 400 bp) could complement these two DNA fingerprinting techniques in detecting changes in brain tumor DNA.
Acknowledgement Dil-Afroze and Anjan Misra are Senior Research Fellows of the University Grants Commission and the Council of Scientific and Industrial Research, Government of India, respectively. We thank Mr Satish Kumar and Mr Mathura Prasad Rai for their technical assistance. This study was partly supported by a core grant from the Department of Biotechnology to the National Institute of Immunology.
References Agurell, E., Li, R., Rannug, U., Norming, U., Tubudait, B., Ramel, C., 1992. Detection of DNA alterations in human bladder tumors
48
Dil-Afroze et al. / Gene 206 (1998) 45–48
by DNA fingerprinting analysis. Cancer Genet. Cytogenet. 60, 53–60. Armour, J.A.L., Jeffreys, A.J., 1992. Biology and applications of human microsatellite loci. Curr. Opin. Genet. Dev. 2, 850–856. Benter, T., Papadopoulos, S., Pape, M., Manns, M., Poliwoda, H., 1995. Optimization and reproducibility of random amplified polymorphic DNA in human. Analyt. Biochem. 230, 92–100. Boland, C.R., 1996. Setting microsatellites free. Nature Med. 2, 972–974. Cheah, Y., Paigen, B., 1996. Mapping new murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR. Mamm. Genome 7, 549–550. Chen, P., Hurst, T., Khoo, S.K., 1991. Detection of somatic DNA alterations in ovarian cancer by DNA fingerprinting analysis. Cancer 67, 1551–1555. de Jong, D., Voetdijk, B.M.H., Kluin-Nelemans, J.C., VanOmmen, G.J.B., Kluin, P.H.M., 1988. Somatic changes in B-Lymphoproliferative disorders (B-LPD) detected by DNA-fingerprinting. Br. J. Cancer 58, 773–775. del Tufo, J.P., Tingey, S.V., 1994. RAPD assay—A novel technique for genetic diagnostics. Meth. Mol. Biol. 28, 237–241. Gutierrez-Adan, A., Cushwa, W.T., Anderson, G.B., Medrano, J.F., 1997. Ovine-specific Y-chromosome RAPD-SCAR marker for embryo sexing. Anim. Genet. 28, 135–138. Ionov, Y., Peinado, M.A., Malkhosyan, S., Shibata, D., Perucho, M., 1993. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363, 558–561.
Joshi, A.R., Sinha, S., Dil-Afroze, Sulaiman, I.M., Banerji, A.K., Hasnain, S.E., 1996. Alterations in brain tumor DNA detected by a fingerprinting probe. Ind. J. Biochem. Biophys. 33, 455–457. McClelland, M., Welsh, J., 1994. DNA fingerprinting by arbitrarily primed PCR. PCR Meth. Appl. 4, S59–S65. Matioli, S.R., de Brito, R.A., 1995. Obtaining genetic markers by using double-stringency PCR with microsatellites and arbitrary primers. Biotechniques 19, 752–756. Matsumura, Y., Tarin, D., 1992. DNA fingerprinting survey of various human tumors and their metastasis. Cancer Res. 52, 2174–2179. Peinado, M.A., Malkhosyan, S., Velazquez, A., Perucho, M., 1992. Isolation and characterization of allelic losses and gains in colorectal tumors by arbitrarily primed polymerase chain reaction. Proc. Natl. Acad. Sci. USA 89, 10065–10069. Thein, S.L., Jeffreys, A.J., Gooi, H.C., Cotter, F., Flint, J., O’connor, N.T.J., Weatherall, D.J., Wainscoat, J.S., 1987. Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br. J. Cancer 55, 353–356. Vogelstein, B., Fearon, E.R., Kern, S.E., Hamilton, S.R., Preisinger, A.C., Nakamura, Y., White, R., 1989. Allelotype of colorectal carcinomas. Science 244, 207–211. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535.
Genetic alterations in brain tumors identified by RAPD analysis Dil-Afroze a,b, Anjan Misra b,1, Irshad M. Sulaiman a, Subrata Sinha b, Chitra Sarkar c, Ashok K. Mahapatra d, Seyed E. Hasnain a,* a Eukaryotic Gene Expression Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India b Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India c Department of Pathology, All India Institute of Medical Sciences, New Delhi, India d Department of Neurosurgery, All India Institute of Medical Sciences, New Delhi, India Received 15 May 1997; received in revised form 23 September 1997; accepted 23 September 1997; Received by J. Wild
Abstract We report the utility of random amplified polymorphic DNA (RAPD) analysis for identifying subtle genomic alterations in ` meningiomas and gliomas by comparing the DNA band profile of tumor vis-a-vis its constitutional counterpart. Twenty out of the 29 decanucleotide GC-rich random primers utilized for the RAPD analysis of meningiomas revealed alteration(s) in the tumor genome. In gliomas, changes were detected by 16 of the 18 primers. While all the seven meningioma samples exihibited alterations in tumor DNA, changes were evident in 21 of the 24 glioma cases. These alterations in tumor DNA included the loss of a normal band, appearance of a new band and amplification of a pre-existing band. Many primers detected more than one alterations in a given tumor. Our approach, which covers the range from 0.4 to 2 kb, besides detecting a significant number of changes in a spectrum of brain tumors, complements existing DNA fingerprinting methods, such as microsatellite mapping ( less than 0.4 kb) and Southern blotting (over 2 kb), for detecting genetic alterations in tumors. © 1997 Elsevier Science B.V. Keywords: Meningioma; Glioma; PCR; Genomic alterations
1. Introduction A number of molecular and cytogenetic methods have been used to identify and characterize genetic alterations in tumors. Of these, the locus non-specific methods of DNA fingerprinting have the potential of identifying novel changes in tumor DNA. Southern hybridization with multi-locus fingerprinting probes has been able to identify changes in malignancies of the gastrointestinal tract, breast, ovary, urinary bladder, meninges, glia and in melanomas ( Thein et al., 1987; de Jong et al., 1988; Chen et al., 1991; Agurell et al., 1992; Matsumura and Tarin, 1992; Joshi et al., 1996). Microsatellite mapping * Corresponding author. Tel: +91 11 6103799/6189622, ext. 301; Fax: +91 11 616-2125; e-mail: [email protected] 1 The first two authors contributed equally to this work. Abbreviations: bp, base pair(s); EtdBr, ethidium bromide; kb, kilobase(s); PCR, polymerase chain reaction; RAPD, random amplified polymorphic DNA. 0378-1119/97/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 5 79 - 9
( Vogelstein et al., 1989; Armour and Jeffreys, 1992; Boland, 1996) with polymorphic single locus probes requires a battery of probes depending on the extent of the chromosomal region to be characterized. The advantages of random amplified polymorphic DNA (RAPD) analysis lie in the technique being locus non-specific and easy identification of the regions of amplification, deletion or rearrangement without prior information about the loci permitting simultaneous screening of multiple regions of the genome ( Welsh and McClelland, 1990; Williams et al., 1990; del Tufo and Tingey, 1994; Benter et al., 1995; Matioli and de Brito, 1995; Cheah and Paigen, 1996). Furthermore, RAPD screening can be performed on a small amount of tumor DNA, and it is relatively easier to clone the altered fragments for further analyses. In the present study, we describe the use of RAPD marker-based DNA fingerprinting for comparing the amplification profiles of tumor DNA in meningiomas and gliomas with their normal leucocyte.
46
Dil-Afroze et al. / Gene 206 (1998) 45–48
2. Experimental and discussion 2.1. PCR amplification with RAPD primers Surgically resected tumor samples were collected from the Neurosurgery Operation Theatre of All India Institute of Medical Sciences, New Delhi, India. Tissue was snap-frozen immediately after surgery and frozen sections cut from the entire tumor tissue. Regions of the tumor having more than 80% neoplastic cells were used for preparing DNA. Leucocytes of same patients were the source of the corresponding normal DNA. 10-mer oligodeoxynucleotide primers of random but definite sequences of 50–80% GC content were procured from GENOSYS, USA. Amplification was carried out in 25-ml reaction volume with varying MgCl concen2 trations (1.5–6 mM ) and annealing temperatures (22–48°C ) using Taq DNA polymerase. The PCR products were resolved by electrophoresis on a 1.5% agarose gel and visualised by ethidium bromide ( EtdBr) staining. Paired samples ( leucocyte and tumor DNA) from seven meningioma and 24 glioma patients were analysed. We scored only those bands as altered where comparison could be made on the basis of intensities of the preceding and succeeding bands in the paired sample. 2.2. RAPD analyses reveal differences between the tumor and the constitutive DNAs Twenty-nine primers ( Table 1) were utilized for screening the meningioma genome for alterations, while 18 primers were used for scoring changes in the gliomas ( Table 2). Of these, nine produced amplification products that were monomorphic (Fig. 1, lanes 1–8) and the remaining 20 detected changes (Fig. 2 lanes 1–8) when the meningioma tumor RAPD profile was compared to that of its normal leucocyte DNA. Two of the 18 primers used for screening gliomas generated a monomorphic profile ( Fig. 1, lanes 9–16), while 16 revealed changes ( Fig. 2, lanes 9–16). A multiband profile was observed for all the primers with the total number of bands per primer varying from 2 to 14 for meningiomas ( Table 1) and from 8 to 22 for gliomas ( Table 2). The size of these bands ranged from 400 bp to 2 kb. With a given primer, all seven meningiomas and 21 out of 24 glioma samples exhibited alteration(s). The alterations exhibited in tumor DNA included the loss of a normal band (Fig. 2, lanes 2, 12 and 16), appearance of a new band (Fig. 2, lanes 4, 6, 8 and 14) and amplification of a preexisting band ( Fig. 2, lanes 8 and 10). Many primers detected more than one such alteration in a given tumor ( Fig. 2, lane 8). RAPD amplified fragments are best resolved between 400 bp and 2 kb, whereas the other fingerprinting methods are best for fragments either larger than 2 kb (Southern blotting) or less than 400 bp (microsatellite
Table 1 Primers utilized for RAPD analysis to score alteration(s) in meningiomas Primer
Sequence (5∞–3∞)
Number of amplification productsa
Polymorphism statusb
2 3 5 7 10 13 14 16 18 26 27 28 32 34 35 36 37 39 41 42 47 48 50 52 54 71 77
CAATGCGTCT AGGATACGTG CGGATAACTG TCCGACGTAT CCATTTACGC TACACTAGCG CATAGCCCTT AGTGAATGCG TTTACGGTGG AAGATAGCGG CCTATCCGTT GATTGCGTTC GTCCTACTCG GTCCTTAGCG GTCCTCAACG CTACTACCGC GAGTCACTCG CGTCGTTACC TGCGCGATCG GCAGGATACG AACGTACGCG CGATGAGCCC CATCCCGAAC CAGGGTCGAC ATCTCCCGGG GCACCCGACG GCACGCCGGA
10 7 11 6 9 10 7 13 10 8 5 6 8 7 5 7 2 7 9 7 9 6 7 14 5 6 12
+ + + + + + − + + + + − − + + + − − + − + + − + + − +
aMean of total number of bands generated with the primer. b‘−’ and ‘+’ denote monomorphic and altered band patterns, respectively, when RAPD profiles for tumor DNA was compared with its constitutive counterpart.
mapping). RAPD analysis covers the range in between and could complement these two DNA fingerprinting techniques. In studies exploiting a similar approach, changes in colorectal cancer were demonstrated by Arbitrarily Primed PCR using primers varying in length from 20 to 28 bases (Peinado et al., 1992; Ionov et al., 1993; McClelland and Welsh, 1994). These changes were demonstrated by autoradiography after PAGE. Our study using 10-mer primers extends the application of this approach to brain tumors besides identifying alterations simply at the level of EtdBr-stained agarose gels. Another possible utility of the procedure is for the development of a simple system for scoring the overall extent of genomic alterations in tumors. Genomic instability is now taken as a measure of the extent of expansion and contraction of repeats at a microsatellite locus (Boland, 1996). Altered RAPD fragments are the manifestation of an unstable genome, and the significance of these in terms of the behavior of a tumor needs to be elucidated. Characterization of such altered fragments would constitute the first step in our understand-
Dil-Afroze et al. / Gene 206 (1998) 45–48
47
Table 2 Primers utilized for RAPD analysis to score alteration(s) in gliomas Primer
Sequence
Number of amplification productsa
Polymorphism statusb
9 24 43 39 59 61 64 70 73 76 82 85 87 88 90 95 96 101
AGAAGCGATG GTTAGTGGCA CTGCGATACC CGCTGTTACC TGCAGCACCG GCCCCTCTTG GGACCGCTAG CGCAGACCTC CCATGGCGCC GCACGGAGGG GCCCCATGCG CACCTGCCGC CGCCAGGAGC CGCGTCAGCC TGGTCGGCGC GCGAATTCCG CGGAATTCGC GGCTGCAGCG
15 9 8 16 18 15 16 12 12 22 17 8 13 11 6 9 11 14
+ + + − + + + + + + + + + − + + + +
aMean of total number of bands generated with the primer. b‘−’ and ‘+’ denotes monomorphic and altered band patterns respectively, when RAPD profiles for tumor DNA was compared with its constitutive counterpart.
Fig. 1. Random amplified polymorphic DNA profile generated by 10-mer primers for paired samples of meningioma and glioma exhibits a monomorphic band pattern. A representative primer-template combination profile generated for a single pair of meningioma with four different primers namely P28, P42, P51, P71 ( lanes 1–8) and glioma samples from two patients with 2 different primers P88, P49 ( lanes 9, 10 and 13, 14 with one pair and lanes 11, 12 and 15, 16 with another pair of sample) are shown. Leucocyte and tumor DNA are indicated by N and T, respectively.
ing of the molecular mechanism leading to the onset of tumorigenesis. Further, based on the information on sequence analysis of these altered fragments, this study could be extended to address the possibility of generating RAPD-derived SCAR markers (Gutierrez-Adan et al., 1997) to detect specific alteration(s) in these forms of brain tumors.
Fig. 2. Polymorphism detected by RAPD primers within the tumor genome. The polymorphic regions within the genomes of meningiomas and gliomas when compared to their normal profile appeared in the form of loss of a normal band ( lanes 2, 12 and 16), appearance of a new band ( lanes 4, 6, 8 and 14) or amplification of a preexisting band ( lanes 8 and 10). A representative RAPD profile using a single pair of meningioma ( lanes 1–8) and glioma ( lanes 9–16) samples with 4 different primers is shown.
3. Conclusions (1) Our study demonstrates that RAPD analysis with 10-mer primers of a random but defined sequence can be used to score subtle alterations in genomes of a large proportion of gliomas and meningiomas. (2) The RAPD marker-based procedure for detecting genetic alterations during tumorigenesis utilizes only nanogram quantities of tissue to begin with and requires simple EtdBr staining for scoring these genomic alterations in brain tumors. (3) RAPD amplified fragments varying from 400 bp to 2 kb covering the range in between Southern blotting ( larger than 2 kb) or microsatellite mapping ( less than 400 bp) could complement these two DNA fingerprinting techniques in detecting changes in brain tumor DNA.
Acknowledgement Dil-Afroze and Anjan Misra are Senior Research Fellows of the University Grants Commission and the Council of Scientific and Industrial Research, Government of India, respectively. We thank Mr Satish Kumar and Mr Mathura Prasad Rai for their technical assistance. This study was partly supported by a core grant from the Department of Biotechnology to the National Institute of Immunology.
References Agurell, E., Li, R., Rannug, U., Norming, U., Tubudait, B., Ramel, C., 1992. Detection of DNA alterations in human bladder tumors
48
Dil-Afroze et al. / Gene 206 (1998) 45–48
by DNA fingerprinting analysis. Cancer Genet. Cytogenet. 60, 53–60. Armour, J.A.L., Jeffreys, A.J., 1992. Biology and applications of human microsatellite loci. Curr. Opin. Genet. Dev. 2, 850–856. Benter, T., Papadopoulos, S., Pape, M., Manns, M., Poliwoda, H., 1995. Optimization and reproducibility of random amplified polymorphic DNA in human. Analyt. Biochem. 230, 92–100. Boland, C.R., 1996. Setting microsatellites free. Nature Med. 2, 972–974. Cheah, Y., Paigen, B., 1996. Mapping new murine polymorphisms detected by random amplified polymorphic DNA (RAPD) PCR. Mamm. Genome 7, 549–550. Chen, P., Hurst, T., Khoo, S.K., 1991. Detection of somatic DNA alterations in ovarian cancer by DNA fingerprinting analysis. Cancer 67, 1551–1555. de Jong, D., Voetdijk, B.M.H., Kluin-Nelemans, J.C., VanOmmen, G.J.B., Kluin, P.H.M., 1988. Somatic changes in B-Lymphoproliferative disorders (B-LPD) detected by DNA-fingerprinting. Br. J. Cancer 58, 773–775. del Tufo, J.P., Tingey, S.V., 1994. RAPD assay—A novel technique for genetic diagnostics. Meth. Mol. Biol. 28, 237–241. Gutierrez-Adan, A., Cushwa, W.T., Anderson, G.B., Medrano, J.F., 1997. Ovine-specific Y-chromosome RAPD-SCAR marker for embryo sexing. Anim. Genet. 28, 135–138. Ionov, Y., Peinado, M.A., Malkhosyan, S., Shibata, D., Perucho, M., 1993. Ubiquitous somatic mutations in simple repeated sequences reveal a new mechanism for colonic carcinogenesis. Nature 363, 558–561.
Joshi, A.R., Sinha, S., Dil-Afroze, Sulaiman, I.M., Banerji, A.K., Hasnain, S.E., 1996. Alterations in brain tumor DNA detected by a fingerprinting probe. Ind. J. Biochem. Biophys. 33, 455–457. McClelland, M., Welsh, J., 1994. DNA fingerprinting by arbitrarily primed PCR. PCR Meth. Appl. 4, S59–S65. Matioli, S.R., de Brito, R.A., 1995. Obtaining genetic markers by using double-stringency PCR with microsatellites and arbitrary primers. Biotechniques 19, 752–756. Matsumura, Y., Tarin, D., 1992. DNA fingerprinting survey of various human tumors and their metastasis. Cancer Res. 52, 2174–2179. Peinado, M.A., Malkhosyan, S., Velazquez, A., Perucho, M., 1992. Isolation and characterization of allelic losses and gains in colorectal tumors by arbitrarily primed polymerase chain reaction. Proc. Natl. Acad. Sci. USA 89, 10065–10069. Thein, S.L., Jeffreys, A.J., Gooi, H.C., Cotter, F., Flint, J., O’connor, N.T.J., Weatherall, D.J., Wainscoat, J.S., 1987. Detection of somatic changes in human cancer DNA by DNA fingerprint analysis. Br. J. Cancer 55, 353–356. Vogelstein, B., Fearon, E.R., Kern, S.E., Hamilton, S.R., Preisinger, A.C., Nakamura, Y., White, R., 1989. Allelotype of colorectal carcinomas. Science 244, 207–211. Welsh, J., McClelland, M., 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18, 7213–7218. Williams, J.G., Kubelik, A.R., Livak, K.J., Rafalski, J.A., Tingey, S.V., 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18, 6531–6535.